Table of conTenTs symposium overview

Document Sample
Table of conTenTs symposium overview Powered By Docstoc
					             Please check the Symposium Program Addendum (to be provided at the meeting) for any changes/updates.

                                                     Table of conTenTs

symposium overview .............................................................................................................................. vi

society organization & committees .................................................................................................... viii

society awards ........................................................................................................................................ xii

call for 2011 award nominations........................................................................................................ xvii

corporate Members, symposium/award sponsors ......................................................................... xviii

General Information .............................................................................................................................. xxi

future Meeting Dates ........................................................................................................................ xxviii

Manchester Grand Hyatt floor Plan

symposium-at-a-Glance ...................................................................................................................... xxxi

symposium Daily Program
   Saturday .................................................................................................................................................1
   Monday ..................................................................................................................................................8
   Tuesday ................................................................................................................................................15
   Wednesday ...........................................................................................................................................21
   Thursday ..............................................................................................................................................28

Poster sessions
   Poster Session 1 ...................................................................................................................................29
   Poster Session 2 ...................................................................................................................................38
   Poster Session 3 ...................................................................................................................................47

exhibit/Poster area floor Plan ...............................................................................................................56

exhibiting companies & Products .........................................................................................................57

exhibitor Workshops ...............................................................................................................................62

Invited speaker abstracts .......................................................................................................................65

Poster session abstracts ..........................................................................................................................85

author/Invited speaker Index ..............................................................................................................276

                                 syMPosIuM overvIeW

  all sessions and activities of the 24th annual symposium of The Protein society will take place
                      at the Manchester Grand Hyatt unless noted otherwise.
saturday, July 31
6:00 pm – 8:30 pm       Meeting registration                                      Douglas Foyer, 1st Level

sunday, august 1
7:30 am – 6:00 pm       Meeting registration                                      Douglas Foyer, 1st Level
8:00 am – 5:00 pm       exhibitor registration                                    Douglas Foyer, 1st Level
9:00 am – 12:00 pm      concurrent Morning symposia 1:                            Douglas Pavilion A, 1st Level
                         Protein Engineering for Solubility
                         and Reconstruction
9:00 am – 12:00 pm      concurrent Morning symposia 2:                            Elizabeth DE, 2nd Level
                         Imaging and Deconstructing Large Complexes
12:15 pm – 1:45 pm      Workshop 1:                                               Elizabeth F, 2nd Level
                         Educator’s Luncheon/Workshop
                         Exploring the RCSB Protein Data Bank
12:15 pm – 1:45 pm      Workshop 2:                                               Elizabeth G, 2nd Level
                         Resume Writing Luncheon/Workshop
2:00 pm – 4:45 pm       concurrent afternoon symposia 3:                          Douglas Pavilion A, 1st Level
                         Protein Stability and Drug Formulation
2:00 pm – 4:45 pm       concurrent afternoon symposia 4:                          Elizabeth DE, 2nd Level
                         Visualizing Proteins in Cells

5:00 pm – 7:10 pm       awards Plenary session 1                                  Douglas Pavilion A, 1st Level
7:30 pm – 9:00 pm       Mentoring committee & education                           Gallery Room, Seaport Courtyard
                        committee – Meeting and Mixer

Monday, august 2
7:30 am – 8:30 am       new Member Welcome breakfast                              Manchester D, 2nd Level
7:30 am – 6:00 pm       Meeting registration                                      Douglas Foyer, 1st Level
8:00 am – 6:45 pm       Poster viewing                                            Exhibit Hall/Douglas Pavilion, 1st Level
9:00 am – 12:00 pm      concurrent Morning symposia 5:                            Douglas Pavilion A, 1st Level
                          Protein Therapeutics and Diagnostics
9:00 am – 12:00 pm      concurrent Morning symposia 6:                            Manchester ABC, 2nd Level
                          Disordered Proteins
10:30 am – 6:30 pm      exhibitor registration                                    Douglas Foyer, 1st Level
12:15 pm – 1:15 pm      Workshop 3:                                               Manchester D, 2nd Level
                          Behind the NIH Study Section Door:
                          A Mock Review Panel
12:15 pm – 1:15 pm      exhibitor Workshop                                        See page 62 for listing.
12:00 pm– 6:45 pm       exhibits open                                             Exhibit Hall/Douglas Pavilion, 1st Level
1:15 pm – 2:45 pm       Poster session 1                                          Exhibit Hall/Douglas Pavilion, 1st Level
2:00 pm – 2:45 pm       refreshments                                              Exhibit Hall/Douglas Pavilion, 1st Level
2:45 pm – 5:20 pm       concurrent afternoon symposia 7:                          Manchester ABC, 2nd Level
                          Protein Folding
2:45 pm – 5:20 pm       concurrent afternoon symposia 8:                          Douglas Pavilion A, 1st Level
                          Membrane Proteins and Receptors
5:30 pm – 6:45 pm       Munch & Mingle reception                                  Exhibit Hall/Douglas Pavilion, 1st Level
7:15 pm                 networking Dinners                                        Douglas Foyer, 1st Level
	                       	 (sign	up	on-site;	first	come,	first	served;	self-pay)
7:30 pm – 9:00 pm       Workshop 4:                                               Manchester D, 2nd Level
                          How to Give A Great Talk

Tuesday, august 3
7:30 am – 5:00 pm     Meeting registration                                      Douglas Foyer, 1st Level
7:30 am – 6:30 pm     exhibitor registration                                    Douglas Foyer, 1st Level
7:30 am – 8:30 am     Members business Meeting                                  Douglas Pavilion A, 1st Level
8:00 am – 6:45 pm     Poster viewing                                            Exhibit Hall/Douglas Pavilion, 1st Level
9:00 am – 11:45 am    Plenary session 1:                                        Douglas Pavilion A, 1st Level
                      fred richards Memorial session
10:00 am –10:45 am    refreshments                                              Exhibit Hall/Douglas Pavilion, 1st Level
10:00 am – 6:45 pm    exhibits open                                             Exhibit Hall/Douglas Pavilion, 1st Level
12:00 pm – 1:00 pm    Workshop 5:                                               Manchester G, 2nd Level
                        Career Panel
12:00 pm – 1:0 pm     exhibitor Workshops                                       See page 63 for listing.
1:15 pm – 2:45 pm     Poster session 2                                          Exhibit Hall/Douglas Pavilion, 1st Level
2:00 pm – 2:45 pm     refreshments                                              Exhibit Hall/Douglas Pavilion, 1st Level
2:45 pm – 5:20 pm     concurrent afternoon symposia 9:                          Douglas Pavilion A, 1st Level
                        Microfluidics and Microarrays
2:45 pm – 5:20 pm     concurrent afternoon symposia 10:                         Manchester ABC, 2nd Level
5:30 pm – 6:45 pm     Munch & Mingle reception                                  Exhibit Hall/Douglas Pavilion, 1st Level
7:15 pm               networking Dinners                                        Douglas Foyer, 1st Level
	                     	 (sign	up	on-site;	first	come,	first	served;	self-pay)
7:30 pm – 9:00 pm     Workshop 6:                                               Manchester D, 2nd Level
                        How To Find A Postdoc Position

Wednesday, august 4
7:30 am – 5:00 pm     Meeting registration                                      Douglas Foyer, 1st Level
7:30 am – 4:00 pm     exhibitor registration                                    Douglas Foyer, 1st Level
8:00 am – 3:15 pm     Poster viewing                                            Exhibit Hall/Douglas Pavilion, 1st Level
9:00 am – 12:15 pm    concurrent Morning symposia 11:                           Douglas Pavilion A, 1st Level
                        Protein Design and Evolution
9:00 am – 12:15 pm    concurrent Morning symposia 12:                           Manchester ABC, 2nd Level
                        Amyloids and Disease
10:00 am – 3:30 pm    exhibits open                                             Exhibit Hall/Douglas Pavilion, 1st Level
10:00 am –10:45 am    refreshments                                              Exhibit Hall/Douglas Pavilion, 1st Level
12:15 pm – 1:45 pm    Graduate Program fair                                     Manchester Foyer, 2nd Level
12:30 pm – 1:30 pm    exhibitor Workshop                                        See page 64 for listing.
12:30 pm – 1:30 pm    Workshop 7:                                               Manchester G, 2nd Level
                        Fundamentals of Good Scientific Writing
	                     	 (special	registration	required)
1:45 pm – 3:15 pm     Poster session 3                                          Exhibit Hall/Douglas Pavilion, 1st Level
1:30 pm – 3:10 pm     undergraduate student research session                    Manchester ABC, 2nd Level
2:00 pm –2:45 pm      refreshments                                              Exhibit Hall/Douglas Pavilion, 1st Level
3:20 pm – 6:20 pm     awards Plenary session 2                                  Manchester ABC, 2nd Level
7:30 pm – 9:30 pm     awards reception & banquet                                Manchester Foyer/ABC, 2nd Level

Thursday, august 5
9:00 am – 12:00 pm    Plenary session 2:                                        Manchester ABC, 2nd Level
                        Macromolecular Machines

          THe ProTeIn socIeTy orGanIZaTIon & coMMITTees

                           socIeTy offIcers anD eXecuTIve councIl

    Daniel raleigh             arthur G. Palmer, III                 Jean baum                  David a. agard
       President                  Past President                 Secretary/Treasurer         University of California,
Stony Brook University,         Columbia University              Rutgers University              San Francisco

Wolfgang baumeister               James bowie                      Zengyi chang                   Jane clarke
Max Planck Institute for      University of California,           Peking University          University of Cambridge
    Biochemistry                   Los Angeles

 Wayne fairbrother             Jacquelyn s. fetrow                   yuji Goto                    carol b. Post
    Genentech                  Wake Forest University             Osaka University              Purdue University

   sheena radford                 Janet l. smith                   brian Matthews               cindy yablonski
  University of Leeds          University of Michigan                   ex officio                  ex officio
                                                                 University of Oregon           Executive	Officer
                                                            Institute of Molecular Biology

            24TH syMPosIuM ProGraM orGanIZers

                                  syMPosIuM orGanIZer
                                         David P. Goldenberg
                                          University of Utah

                            ProGraM PlannInG coMMITTee
          James M. berger                                                 Karyn o’neil
   University of California, Berkeley                           Johnson & Johnson Internal Ventures

          Ineke braakman                                                   carol b. Post
          Utrecht University                                             Purdue University

         stephen J. Demarest                                             Peter oelschlaeger
             Biogen Idec                                  California State Polytechnic University, Pomona

                          2010 absTracT revIeW coMMITTee
        John osterhout, Chair                                                 sen liu
        Angelo State University                                University of California, San Francisco
         akhilesh bhambhani                                            Dahabada lopes
          Merck & Co., Inc.                                 David Geffen School of Medicine at UCLA
       alexandre de brevern                                                liang yu
 INSERM, Universite Paris Diderot INTS                             Genome Institute of Singapore
          andrew Hausrath                                              Weiya Douglas lu
         University of Arizona                        School of Pharmacy, University of California, San Diego
              Pavan Jutur                                               a. scott Muerhoff
     University of Nebraska-Lincoln
                                                                        Abbott Laboratories
            Thijs Kaper
                                                                        alison H. varghese
    Genencor, a Division of Danisco
                                                                            Pfizer,	Inc.
              Huiyuan li
David Geffen School of Medicine at UCLA

                               MeeTInG ManaGeMenT
                      FASEB, Office of Scientific Meetings and Conferences
                                   Marcella Jackson, Director
                                 Roya Jaseb, Meeting Manager
                               Danielle King, Meeting Assistant
                                Janet Kearney, Exhibit Manager
                                Joni Friedman, Exhibit Assistant
                                    Josie Leftwich, Registrar

                                  socIeTy coMMITTees

         Education Committee                            bertrand Garcia-Moreno
                                                        Johns Hopkins University
           David bevan, Chair
       Virginia Polytechnic Institute                          blake Hill
                 ellis bell                             Johns Hopkins University
         University of Richmond
                                                       Judith Klein-seetharaman
              lisa Gentile                        University of Pittsburgh Medical Center
         University of Richmond
                                                            Kevin J. lumb
              Mary Konkle                              Merck Research Laboratories
             Trinity University
                                                             adrian Whitty
            angela lombardi                                 Boston University
      Universitá di Napoli Federico II
                                                            Derek Woolfson
             John osterhout                                University of Bristol
          Angelo State University
                                                          arthur G. Palmer, III
        cindy yablonski, ex officio                        Columbia University
            Executive	Officer
                                                       cindy yablonski, ex officio
                                                           Executive	Officer
          Finance Committee
            Jean baum, Chair                             Mentoring Committee
            Rutgers University
                                                           blake Hill, Chair
             Daniel raleigh                             Johns Hopkins University
     Stony Brook University, SUNNY
                                                           Matthew cordes
          arthur G. Palmer, III                           University of Arizona
           Columbia University
                                                          Giovanna Ghirlanda
          Gerald Hazelbauer                              Arizona State University
     University of Missouri-Columbia
                                                            Patricia Jennings
        brian Matthews, ex officio                  University of California, San Diego
          University of Oregon
                                                       Judith Klein-seetharaman
        cindy yablonski, ex officio               University of Pittsburgh Medical Center
            Executive	Officer
                                                            Kevin J. lumb
                                                       Merck Research Laboratories
        Membership Committee                                Janel r. Mclean
       Daniel raleigh, Interim Chair                       Vanderbilt University
State University of New York at Stony Brook
                                                            John a. Mclean
               David bevan                                 Vanderbilt University
       Virginia Polytechnic Institute
                                                           Igor Mochalkin
           Jennifer r. cochran                        EMD Serono Research Institute
            Stanford University
                                                             olve Peersen
            Wendy cornell                               Colorado State University
       Merck Research Laboratories
                                                             Peter schubert
           stephen J. Demarest                        University of British Columbia
               Biogen Idec

     andrew c. vendel                            Publication Committee
    Eli Lilly and Company
                                                  James bowie, Chair
  cindy yablonski, ex officio              University of California-Los Angeles
      Executive	Officer
                                                       Jean baum
                                                    Rutgers University
  Nominating Committee
                                                       Daniel raleigh
     Jane clarke, Chair                 State University of New York at Stony Brook
   University of Cambridge
                                                     lila M. Gierasch
       angel e. Garcia                     University of Massachusetts, Amherst
Rensselaer Polytechnic Institute
                                                  Gerald Hazelbauer
       lisa M. Gloss                         University of Missouri-Columbia
 Washington State University
                                                  arthur G. Palmer, III
  cindy yablonski, ex officio                      Columbia University
      Executive	Officer
                                                       Jeffery Kelly
                                               The Scripps Research Institute
                                                   c. robert Matthews
                                          University of Massachusetts Med School
                                                brian Matthews, ex officio
                                                  University of Oregon
                                                cindy yablonski, ex officio
                                                    Executive	Officer

                                THe ProTeIn socIeTy aWarDs

            The following awards will be conferred at the 24th Annual Symposium of The Protein Society
                      in San Diego, CA at the Awards Plenary Session 1 on Sunday, August 1.

                                               The Carl Brändén Award
                        The Carl Brändén Award, sponsored by Rigaku Corporation, is given to an outstanding protein
                        scientist who has also made exceptional contributions in the areas of education and/or service to the
                        science. The award is granted annually – alternating between the U.S. and European meetings.
                       The 2010 award will be presented to Dr. Nobuhiro Go (RIKEN Harima Institute, Japan) for his
                       scientific achievements, including the establishment of a lattice model of proteins, now well-known
                       as the Go model. Dr. Go has also contributed markedly to the NMR distance geometry analysis as
                       well as the analysis of protein dynamics. His current work in protein biophysics, protein physical
                       chemistry	and	computer	science	has	brought	important	physical	principles	to	the	field.	Dr.	Go	is	
                       widely considered to be a pioneer in the theoretical study of protein folding principles, with many
     Nobuhiro Go     younger protein scientists owing a great deal to his ideas. In 2001, Dr. Go was one of the founders
of	the	Protein	Science	Society	of	Japan,	which	has	significantly	promoted	the	globalization	of	protein	science.
Previous	award	recipients	include:		Bruce	Alberts	(2009),	Howard	Schachman	(2008),	Dr.	Lubert	Stryer	(2007).

                                          The Christian B. Anfinsen Award
                        The	Christian	B.	Anfinsen	Award,	sponsored by The Aviv Family Foundation,	recognizes	significant	
                        technical	achievements	in	the	field	of	protein	science.		
                        The 2010 award will be presented to Dr. Yoshinori Fujiyoshi (Kyoto University) for his
                        groundbreaking work in the field of membrane protein structure and for bringing electron
                        crystallography to a new level by developing innovative cryoelectron microscopes. Dr. Fujiyoshi’s
                        innovative work has made it possible to use electron microscopy to solve the 3D structures of a
                        variety of channels, receptors and other membrane proteins. His current research efforts are on:
                        bacteriorhodopsin; the plant light-harvesting complex; the structure of several members of the
                        aquaporin	family;	and	the	acetylcholine	receptor.	Dr.	Fujiyoshi’s	contributions	in	the	field	of	electron	
 Yoshinori Fujiyoshi    cryomicroscopy	have	set	the	gold	standard	for	others	in	the	field	to	emulate.
Previous	award	recipients	include:		Wayne	Hubbell	(2009),	Carol	Robinson	(2008),	Carl	Frieden	(2007),	John	Yates,	III	
(2006),	Matthias	Mann	(2005),	Meir	Wilchek	(2004),	Ada	Yonath	(2003),	Roger	Tsien	(2002),	Martin	Karplus	(2001),	Stephen	
Benkovic	(2000),	Alan	Fersht	(1999),	James	Wells	(1998),	Wayne	Hendrickson	(1997),	and	Donald	Hunt	(1996).	

                                           The Emil Thomas Kaiser Award
                        The Emil Thomas Kaiser Award, sponsored by The Protein Society, recognizes a recent and highly
                        significant	contribution	to	the	application	of	chemistry	to	the	study	of	proteins.		
                         The 2010 award will be presented to Dr. Suzanne Walker (Harvard Medical School) for her
                         achievements in the structure and function of enzymatic machinery in an effort to change the
                         way scholars think about natural product antibiotics such as ramoplanin and moenomycin
                         and how they work to block bacterial cell wall assembly. Dr. Walker’s studies have included
                         the determination of the gene clusters involved in the biosynthesis of moenomycin, the detailed
                         enzymological dissection of its functional components, and the mechanisms of several natural
                         products that inhibit the biosynthetic pathway. Currently, her lab is investigating the biosynthesis
   Suzanne Walker        of teichoic acid and anionic carbohydrate based polymers that are covalently attached to the
peptidoglycan matrix of many gram positive bacteria. Dr. Walker is a world-class organic chemist/chemical biologist in
the best research tradition of the late Professor Emil Thomas Kaiser.

Previous	award	recipients	include:		Donald	Hilvert	(2009),	JoAnne	Stubbe	(2008),	Michael	Marletta	(2007),	Barbara	
Imperiali	(2006),	Ronald	Raines	(2005),	Homme	Hellinga	(2004),	Michael	Hecht	(2003),	and	Steve	Kent	(2002).	The	award	
was	previously	named	the	Amgen	Lectureship,	under	which	recipients	included	Robert	Sauer	(2001),	David	Eisenberg	
(2000),	Randy	Schekman	(1999),	M.	Yoshida	(1998),	Stan	Prusiner	(1997),	T.	Tsukihara	and	S.	Yoshikawa	(1996),	and	
Jane	and	David	Richardson	(1995).	

            The following awards will be conferred at the 24th Annual Symposium of The Protein Society
                     in San Diego, CA at the Awards Plenary Session 2 on Wednesday, August 4.

                                    The Irving Sigal Young Investigator Award
The Irving Sigal Young Investigator Award, sponsored by Merck Research Laboratories,	recognizes	a	significant	contribution	
to the study of proteins by a scientist who is in the early stages of an independent career and, generally, not more than 40
years of age at the time of the award.
                        The 2009 award will be presented to Dr. Virginia Cornish (Columbia University) for combining
                        modern techniques in synthetic organic chemistry and molecular biology that manipulate
                        nature’s machineries to carry out new chemistry. Dr. Cornish’s laboratory is pioneering
                        chemical approaches to cell engineering—moving beyond advances in the last century for the
                        synthetic manipulation of biomolecules in vitro and creating conceptually new approaches for the
                        modification	of	biomolecules	in	a	living	cell.		The	main	project	in	her	laboratory	is	a	“chemical	
                        complementation” system for the directed evolution of enzymes with new properties. This project
                        addresses	the	major	bottleneck	in	the	directed	evolution	field—how	to	select	for	enzyme	catalysis	
                        of a wide variety of chemical transformations. Dr. Cornish has emerged as one of the top young
  Virginia Cornish      faculty in the world in the area of chemical biology—her research being noted for its innovation
                        and creativity as recognized by numerous awards.

                        The 2010 award will be presented to Dr. Charalampos Kalodimos (Rutgers University) for his
                        landmark study of the mechanism of protein secretion. Dr. Kalodimos was able to solve the
                        structure of the SecA protein in complex with a signal sequence using advanced NMR and isotope
                        labeling	techniques.	His	lab	has	recently	shed	new	light	on	“dynamic	allostery.”	Dr.	Kalodimos	is	
                        a rising star who possesses tremendous energy, vision and passion.
                        Previous	recipients	include:		Jamie	Doudna	Cate	(2008),	Benjamin	Cravatt,	III	(2007),	Vijay	Pande	
                        (2006),		Thomas	Muir	(2005),	Erin	O’Shea	and	Jonathan	Weissman	(2004),	Yigong	Shi	(2003),	
                        Carolyn	Bertozzi	(2002),	Kevan	Shokat	(2001),	David	Baker	(2000),	Jeffery	Kelly	(1999),	Nikola	
                        Pavletich	(1998),	John	Kuriyan	(1997),	Michael	Summers	(1996),	Stuart	Schreiber	(1995),	Peter	
    Charalampos         Kim	(1994),	Ad	Bax	and	Marius	Clore	(1993),	Peter	Schultz	(1992),	Carl	Pabo	(1991),	Rachel	
     Kalodimos          Klevit	(1990),	and	William	DeGrado	(1989).

                                             The Stein and Moore Award
                        The Stein and Moore Award, sponsored by The Protein Society, and named for Nobel Laureates Dr.
                        William Stein and Dr. Stanford Moore, venerates their contribution to understanding the connection
                        between chemical structure and catalytic activity of the active center of the ribonuclease molecule.
                        To that end, recipients of the Stein and Moore Award are recognized for their contributions to the
                        highest level of the study of proteins.
                        The 2010 award will be presented to Dr. Peter Wright (The Scripps Research Institute) for his
                        studies in DNA bending, his work on specific transcription factor complexes, and his development
                        of new NMR methods along with his investigation of conformational fluctuations and dynamics in
     Peter Wright
                        the catalytic cycle of dihydrofolate reductase. Dr. Wright’s current research seeks to understand the
                        intrinsically disordered regions of proteins in order to move beyond structure determination and the
description of the dynamics of the system. Regarded by colleagues as a leader in both structural biology and protein science,
Dr. Wright is especially admired for the way he has selected protein science problems (such as how folding is coupled to
biological	function	and	catalysis)	whose	solutions	will	have	broad	significance	and	applicability	for	decades	to	come.

Previous	award	recipients	(sponsored	by	The	Protein	Society)	include:		Peter	Walter	(2009).		Previous	award	recipients	
(sponsored	by	The	Merck	Company	Foundation)	include:	Susan	Lindquist	(2008),	Paul	Schimmel	(2007),	F.	Ulrich	Hartl	
and	Arthur	 Horwich	 (2006),	Avram	 Hershko	 and	Alexander	 Varshavsky	 (2005),	 Wolfgang	 Baumeister	 (2004),	 Chris	
Dobson	(2003),	Paul	Sigler	(2002),	Alan	Fersht	(2001),	Brian	Matthews	(2000),	Mo	Cleland	(1999),	David	Davies	(1998),	
Mildred	Cohn	(1997),	David	Eisenberg	(1996),	Harold	Scheraga	(1995),	Michael	Rossman	(1994),	Walter	Kauzmann	
(1993),	Robert	Baldwin	(1992),	Russell	Doolittle	(1991),	Kurt	Wüthrich	(1990),	Hans	Neurath	(1989),	Fred	Richards	
(1988),	and	Emil	Smith	(1987).	

                                      The Dorothy Crowfoot Hodgkin Award
                        The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is granted in recognition of
                        exceptional	 contributions	 in	 protein	 science,	 which	 profoundly	 influence	 our	 understanding	 of	
                        biology. The award is granted annually – alternating between the U.S. and European meetings.
                       The 2010 Award will be presented to Dr. Lila Gierasch (University of Massachusetts, Amherst)
                       for her application of biophysical methods to interrogate biological systems. Dr. Gierasch’s
                       research	has	had	a	major	impact	in	fields	spanning	sequence-structure	relationships,	protein	folding	
                       and	 aggregation,	 the	 pioneering	 application	 of	 novel	 biophysical	 analyses	 (principally	 NMR),	
                       molecular recognition, and cooperativity in molecular machines and protein secretion. Her most
                       recent	research	focuses	on	the	chaperone-mediated	folding	process,	how	a	β-sheet	“clam”	protein	
    Lila Gierasch      is folded, and how to monitor protein folding in a living cell and compare it with in vitro folding.
Dr. Gierasch’s research is considered to be insightful and visionary by her peers.
Previous	award	recipients	include:		Janet	Thornton	(2009),	Douglas	Rees	(2008),	Leemor	Joshua-Tor	(2007).

            The following award will be conferred at the 25th Annual Symposium of The Protein Society
                                    in Boston, MA to be held July 23-27, 2011.

                                              The Hans Neurath Award
                        The Hans Neurath Award, sponsored by The Hans Neurath Foundation, recognizes an individual
                        who	has	made	a	recent	contribution	of	unusual	merit	to	basic	research	in	the	field	of	protein	science,	
                        including but not restricted to the chemistry, design, folding, structure, or biological function of
                          The 2010 award will be presented to Dr. Wendell Lim (University of California, San Francisco-
                          HHMI) in 2011 for his studies encompassing a variety of techniques for understanding protein
                          function. Dr. Lim’s research has set the stage for a new future in biology, which will enable
                          researchers to engineer new signaling systems with potential implications for medicine and other
                          biological applications. He has made seminal contributions that are central to a deep molecular
      Wendell Lim         understanding of cellular signaling pathways as well as illuminating the molecular logic of signal-
transduction networks. At present, Dr. Lim’s research is aimed at linking together multiple regulatory modules to create
cells that will be able to polarize and migrate in response to novels sets of signals. Dr. Lim is considered to be one of the
foremost scientists working on the molecular understanding of signal transduction systems, with a remarkable range of
scientific	abilities	in	areas	such	as	structural	analysis,	biochemistry,	cell	biology	and	mathematical	modeling	of	cellular	
Previous	award	recipients	include:		William	Eaton	(2009),	Robert	Stroud	(2008),	Robert	Sauer	(2007),	Christopher	Dobson	
(2006),	Roderick	MacKinnon	(2005),	Carlos	Bustamante	(2004),	James	Wells	(2003),	Ad	Bax	(2002),	Arthur	Horwich	
(2001),	Janet	Thornton	(2000),	Peter	Kim	(1999),	and	Ken	Dill	(1998).

    The Protein Science young Investigator Travel Grants Program is sponsored by Wiley-blackwell.

            The Protein society finn Wold Travel awards is supported by generous support
                        from members and benefactors of The Protein society.

                       The Protein society acknowledges the 2010 awardees of
 The Protein Science Young Investigator Travel Grants and The Protein Society Finn Wold Travel Awards

                  Mihai azoitei                                      Davin M. Henderson
             University of Washington                          University of Minnesota Twin Cities
             somayesadat badieyan                                       Joanna Hobbs
                 Virginia Tech                                University of Waikato, New Zealand
               brittany a. barry                                             chen Jin
             Montana State University                      National Institute of Natural Sciences, Japan
                    angelo bella                                      Ivan Korendovych
      University of Leicester, United Kingdom                      University of Pennsylvania
             lyra (chia-yin) chang                                      beena Krishnam
             University of Michigan                           University of Massachusetts, Amherst
                 Izhack cherney                                              Xin li
               Princeton University                                   Ohio State University
                amit choudhary                                             Qing lin
         University of Wisconsin-Madison                          University at Buffalo, SUNY
                 Prajwal ciryam                                             sen liu
Northwestern University Feinberg School of Medicine          University of California, San Francisco
             bruno emanuel correia                                         samir Maji
             University of Washington                                   IIT Bombay, India
                armando del rio                                      anna louise Mallam
               Columbia University                                 University of Texas, Austin
                   ann Du                                                 seema Mittal
           Monash University, Australia                    University of Massachusetts Medical School
                    linda foit                                      Issur Moheshwarnath
           University of Michigan/HHMI                          University of Sherbrooke, Canada
                  Gabriel frank                                         sara Jean Moore
            National Institutes of Health                              Stanford University
                elka r. Georgieve                                        Keunwan Park
                Cornell University                                       KAIST, Korea
                 anupam Goel                                         Matthew ranaghan
             Montana State University                               University of Connecticut
                  ankit Gupta                                           Debashish sahu
              University of Rochester                          University of Texas Medical Branch
                  linnea Hedin                                         cristina sandoval
           Stockholm University, Sweden                         University of Colorado at Boulder
         Dahabada Helena Jose lopes                                   Jennifer e. seedorf
       University of California, Los Angeles                        Johns Hopkins University

              lorenza sisinni                                                robert Wells
          University of Padua, Italy                                   Johns Hopkins University
    nagasundarapandian soundrarajan                                       remigiusz Worch
    Pusan National University, South Korea                                BIOTEC, Germany
                Juliana The                                               Jason Ho-lun yau
            University of Arizona                                  Hospital for Sick Children, Toronto
             David Tulumello                                             Hui-Min Zhang
            University of Toronto                          National High Magnetic Field Lab, Florida State
            James van Deventer
      California Institute of Technology

The faseb Minority access to research careers (Marc) Program is supported by a grant from the
          national Institute of General Medical sciences, national Institutes of Health.

                The Protein society acknowledges the 2010 Marc Travel awardees.
                      Please refer to the addendum for additional awardees.
                                           (as of June 18, 2010)
                                         armando Del rio, Ph.D.
                                           Postdoctoral Research
                                            Biological Sciences
                                       Columbia University, New York

                                        lanyn Perez Taliaferro
                                            Graduate Student
                                University of Maryland Baltimore County

  call for noMInaTIons for THe ProTeIn socIeTy’s 2011 aWarDs

2011 aWarDs noMInaTIons
The Protein Society is seeking nominations for the 2011 Society Awards. The Brändén and Hodgkin awards will be
presented at the IX European Symposium of The Protein Society in Stockholm, Sweden, May 22-26, 2011. The Neurath,
Anfinsen,	Stein	and	Moore,	Kaiser,	and	Sigal	awards	will	be	presented	at	the	25th	Annual	Symposium	of	The	Protein	
Society in Boston, Massachusetts, July 23-27, 2011. Recipients will present plenary lectures at the symposia on the
structure	and	function	of	protein	science	as	it	relates	to	their	field	of	study.		The	deadline for submitting complete award
nominations packages for the 2011 Awards cycle will be october 1, 2010. The Society requires that all nominees be
living at the time of an award nomination.

HoW To noMInaTe
Nominations should be e-mailed by the October 1, 2010 deadline to: Please note that the Society
will only accept submissions in PDF format—preferably one PDF per single, complete award nomination package. For
more information, please visit Also note that previous nominations are
automatically carried over for two years; subsequent renominations must be submitted as indicated above. If you have
questions,	please	contact	Cindy	A.	Yablonski,	Executive	Officer,	The	Protein	Society	at
or	(301)	634-7277,	(800)	99-AMINO.		We	look	forward	to	receiving	your	nominations.

                                               The carl brändén award
In the tradition of the late Carl Brändén, pioneer in structural biology, co-author of the seminal text, Introduction to
Protein Structure, and leader of the world-class synchrotron facility at Grenoble, the Carl Brändén Award, sponsored by
Rigaku Corporation, is given to an outstanding protein scientist who has also made exceptional contributions in the areas
of education and/or service to the science.

                                       The Dorothy crowfoot Hodgkin award
The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is granted in recognition of exceptional contributions
in	protein	science,	which	profoundly	influence	our	understanding	of	biology.	

                                                 The stein and Moore award
The Stein and Moore Award, sponsored by The Protein Society and named for Nobel Laureates Dr. William Stein and Dr.
Stanford Moore, venerates their contribution to understanding the connection between chemical structure and catalytic
activity of the active center of the ribonuclease molecule. To that end, recipients of the Stein & Moore award are recognized
for their contributions to the highest level of the study of proteins.

                                                 The Hans neurath award
The Hans Neurath Award, sponsored by the Hans Neurath Foundation, recognizes an individual who has made a recent
contribution	of	unusual	merit	to	basic	research	in	the	field	of	protein	science,	including	but	not	restricted	to	the	chemistry,	
design, folding, structure, or biological function or proteins.

                                         The Christian B. Anfinsen Award
The	Christian	B.	Anfinsen	Award,	sponsored	by	The	Aviv	Family	Foundation,	recognizes	significant	technical	achievements	
in	the	field	of	protein	science.

                                           The emil Thomas Kaiser award
The	Emil	Thomas	Kaiser	Award,	sponsored	by	The	Protein	Society,	recognizes	a	recent,	highly	significant	contribution	
in applying chemistry to the study of proteins.

                                       The Irving sigal young Investigator award
The Irving Sigal Young Investigator Award, sponsored by Merck Research Laboratories, is an award that recognizes an
important contribution to the study of proteins by a scientist who is in the early stages of an independent career. Candidates
are generally not more than 40 years of age.

      corPoraTe MeMbers anD syMPosIuM/aWarD sPonsors

                                                 (as of June 15, 2010)

                                          2010 Corporate Members
          The Protein society acknowledges the continued generous support of its corporate Members.

               Gold corporate Members                                        Silver corporate Members
                      Genentech                                                 Aviv Biomedical, Inc.
          Pierce	now	sold	as	Thermo	Scientific                                          Jasco
                                                                                  Wyatt Technology

                                           2010 Meeting Sponsors
                 The Protein society acknowledges the generous support of its meeting sponsors
                                           Bristol-Myers Squibb Company
                                               Eli Lilly and Company*
                                                New England BioLabs
                                                   NIGMS, NIH**
*Eli Lilly and Company sponsors the Student Poster Awards. This activity is supported by a contribution from Lilly USA,
LLC. For further information concerning Lilly grant funding visit
**The 24th Symposium is supported in part by Award Number R13GM095262 from the National Institute of General
Medical Sciences (NIGMS); support does not necessarily represent the official views of the NIGMS or the National
Institutes of Health.

                                              2010 Award Sponsors
             The Protein society acknowledges the generous support of the sponsors who have made
                         possible the following awards for excellence in protein science
                                             The carl brändén award
                                          sponsored by Rigaku Corporation
                                      The Dorothy crowfoot Hodgkin award
                                             sponsored by Genentech
                                            The stein and Moore award
                                          sponsored by The Protein Society
                                             The Hans neurath award
                                     sponsored by The Hans Neurath Foundation
                                         The Christian B. Anfinsen Award
                                      sponsored by The Aviv Family Foundation
                                          The emil Thomas Kaiser award
                                          sponsored by The Protein Society
                                    The Irving sigal young Investigator award
                                     sponsored by Merck Research Laboratories

                             The 2010 Finn Wold Travel Award Contributors
                The Protein society acknowledges these individuals for their support of the
                                     2010 finn Wold Travel awards.
                Mrs. finn Wold                                         Dr. Daniel P. raleigh
                                                                   Stony Brook University, SUNY
             Dr. linda chapman
        University of Missouri-Columbia                                 Dr. George D. rose
                                                                      Johns Hopkins University
             Dr. anne Gershenson
              Brandeis University                                       Dr. Madeline a. shea
                                                            University of Iowa Carver College of Medicine
                 Dr. blake Hill
            Johns Hopkins University                                   Dr. charles W. sokolik
                                                                         Denison University
               Dr. Peter c. Kahn
               Rutgers University                                    Dr. cynthia v. stauffacher
                                                                          Purdue University
            Dr. Takamitsu Kohzuma
                Ibaraki University                                       Dr. Donald steiner
                                                                        University of Chicago
           Dr. c. James McKnight
     Boston University School of Medicine                                Dr. Marc s. Wold
                                                                         University of Iowa
             Dr. arthur G. Palmer
              Columbia University                                      Dr. Beulah M. Woodfin
                                                                      University of New Mexico
                Dr. Jack Preiss
            Michigan State University

                                  2010 Education Committee Contributors
                The Protein society acknowledges these individuals for their support of the
                              education committee activities of the society.

               Dr. David r. bevan                                        Dr. Peter c. Kahn
Virginia Polytechnic Institute and State University                      Rutgers University
           Dr. William f. DeGrado                                     Dr. Takamitsu Kohzuma
   University of Pennsylvania Medical Center                              Ibaraki University
            Dr. Julie forman-Kay                                       Dr. susan Marqusee
       Hospital for Sick Children, Toronto                        University of California, Berkeley
             Dr. anne Gershenson                                     Dr. cynthia v. stauffacher
              Brandeis University                                         Purdue University
            Dr. Mary G. Hamilton                                         Dr. lisa M. unico
       Fordham College at Lincoln Center                                 Edinboro University
                 Dr. blake Hill                                    Dr. JoAnn L. Whitefleet-Smith
            Johns Hopkins University                               Worcester Polytechnic Institute
               Dr. James c. Hu
             Texas A&M University

                                 2010 Mentoring Committee Contributors
                The Protein society acknowledges these individuals for their support of the
                             Mentoring committee activities of the society.

            Dr. William f. DeGrado                                   Dr. cynthia v. stauffacher
    University of Pennsylvania Medical Center                             Purdue University
                 Dr. blake Hill                                          Dr. Tianzhu Zang
            Johns Hopkins University                                   Northeastern University
             Dr. cynthia s. randall

                                 2010 General Contributors to the Society
The Protein society acknowledges these individuals for their support of the society’s general activities,
                          including public policy and outreach programs.
           Dr. leonard J. banaszak                                    Dr. Norma J. Greenfield
       University of Minnesota Twin Cities                               Highland Park, NJ
           Dr. esther M. G. breslow                                        Dr. blake Hill
               Cornell University                                     Johns Hopkins University
              Dr. David S. Cafiso                                      Dr. William c. Kenney
              University of Virginia                                     Thousand Oak, CA
               Dr. carolyn cohen                                        Dr. lilia Koriazova
               Brandeis University                                     Kyowa Kirin California
               Dr. alan Davidson                                    Dr. Marie-claire McKenna
              University of Toronto                                Siemens Healthcare Diagnostics
              Dr. David r. Davies                                       Dr. evan T. Powers
                  NIDDK, NIH                                        The Scripps Research Institute
               Dr. H. Jane Dyson                                       Dr. cynthia s. randall
          The Scripps Research Institute                                   Sanofi-Aventis
               Dr. John Garaveli                                     Dr. cynthia v. stauffacher
              The RESID Database                                          Purdue University
             Dr. Jenny P. Glusker                                        Dr. Donald steiner
            Fox Chase Cancer Center                                     University of Chicago

                                         General InforMaTIon

reGIsTraTIon                                                          new Member fees
                                                                      Registration & New Membership                             $695
exhibitor registration – Douglas Pavilion foyer,                      			(Includes	banquet	ticket)
1st level                                                             Postdoctoral Registration & New Membership                $460*
All exhibitors must register. Exhibitors are granted access           Student Registration & New Membership                     $340*
to	 the	 exhibit	 floor	 during	 set-up/tear-down	 as	 well	 as	      One-Day Graduate Students and New Membership              $280*
one hour before the exhibits open and one hour after the
exhibits close on exhibit days. Exhibitor badges are strictly         nonmember fee
for exhibit personnel working in your company’s booth.                Nonmember Registration                                    $685**
Exhibitor	badges	do	not	permit	entry	to	scientific	sessions.	         Undergraduate Registration                                $100**
Each representative of an exhibiting company must always              ** Registration Fee does not include Awards Banquet
wear	the	official	badge	while	in	the	exhibit	area.		Exhibitors	          ticket
will receive 6 badges per 10 x10 booth. Additional badges             ** Registration Fee does not include Membership or
may be purchased for $20.00 per badge. Lost or misplaced                 Awards Banquet ticket
badges may be purchased for $10.00 per badge.                         Tickets	(based	upon	availability)
exhibitor registration will be open during the                        Banquet Ticket                                            $100
following days and hours:                                             Educators Luncheon                                        $35
Sunday, August 1 – 8:00 am – 5:00 pm                                  Resume Writing Luncheon                                   $35
Monday, August 2 – 10:30 am – 6:30 pm
                                                                      student and Postdoctoral registration
Tuesday, August 3 – 7:30 am – 6:30 pm
                                                                      Any Protein Society student member or regularly matriculated
Wednesday, August 4 – 7:30 am – 4:00 pm
                                                                      student working toward a degree in one of the biomedical
exhibit installation dates and hours:                                 sciences is eligible for a reduced registration fee. Hospital
Sunday, August 1 – 12:00 pm – 5:00 pm                                 residents/interns and laboratory technicians do not qualify
                                                                      as students. Postdoctoral fellows may also register at a
exhibit tear down dates and hours:                                    discounted rate. For students and postdoctoral fellows please
Wednesday, August 4 – 3:30 pm – 8:00 pm                               provide	one	of	the	following	identifications:	a	certificate	of		
                                                                      eligibility,	a	letter	signed	by	the	department	head	on	official	
Meeting registration – Douglas Pavilion foyer,                        stationery, or a student ID card for on-site registration.
1st level
The registration area is located on the 1st Level and will            Press registration
open at 6:00 pm	on	Saturday,	July	31	(refer	to	hours	below).	         Complimentary Press badges will be issued on-site to
Registration	includes	admission	to	all	scientific	and	poster	         members of the working press and freelance writers. Press
sessions, exhibits, one copy of the Program/Abstract book             credentials	 or	 an	 official	 letter	 from	 an	 editor	 bearing	 a	
and one tote bag. Registration does not include any meals.            letter of assignment must be provided to register as press.
                                                                      Representatives	 of	 allied	 fields	 (public	 relations,	 public	
Days/Hours                                                            information,	 public	 affairs)	 are	 not	 qualified	 for	 press	
Saturday, July 31                          6:00 pm – 8:30 pm          registration and must register as non-members.
Sunday, August 1                           7:30 am – 6:00 pm
Monday, August 2                           7:30 am – 6:00 pm          registration refunds/cancellations
Tuesday, August 3                          7:30 am – 5:00 pm          Refunds for registration must have been made in writing
Wednesday, August 4                        7:30 am – 5:00 pm          and postmarked by July 1, 2010. Badge and receipt must
                                                                      accompany	 the	 request	 (except	 for	 non-US	 registrants).	
Member fees                                                           Refunds cannot be made if badges are not received by
Member	(includes	banquet	ticket)	                       $475          the	Society	Office,	or	if	postmarked	after	this	date.	A	$40	
Emeritus                                                $290          cancellation fee will be deducted from all registration
Postdoctoral Member                                     $340*         payment refunds. Attendees that cancel AFTER July 1 will
Student Member                                          $280*         not receive a refund.
One-Day Graduate Students                               $220*

auDIo/vIsual                                                             being	a	means	of	identification,	the	name	badge	is	required	
The	audio/visual	office	is	located	in	the	Oxford	Room	on	                for	 admission	 to	 scientific	 sessions	 and	 exhibits.	 Each	
the	3rd	Level.		The	office	will	be	open:                                 registrant will receive one copy of the Program/Abstract
                                                                         book and one tote bag. Please take your pick-up tickets to
Saturday, July 31 – 4:30 pm – 8:30 pm
                                                                         the	“Publications”	counter	in	the	registration	area.
Sunday, August 1 – Wednesday, August 4 – 7:30 am – 5:00 pm
Thursday, August 5 –7:30am – 12:00pm                                     busIness cenTer
                                                                         The business center is located on the 1st Level and is open
aWarDs recePTIon & banQueT
                                                                         daily. Services offered include email, photocopying and
Wednesday, august 4, 7:30 pm                                             faxing.
The Awards Reception will begin at 7:30 pm in the Manchester,
Foyer on the 2nd Level with the banquet following in                     cHIlD care
Manchester ABC. The Awards Banquet will recognize the                    The Protein Society does not provide formal child
following award winners: The Carl Brändén Awardee; The                   care at the Symposium. You may contact the hotel
Christian	B.	Anfinsen	Awardee;	The	Emil	Thomas	Kaiser	                   concierge for recommendations or further information at
Awardee; The Irving Sigal Young Investigator Award 2009                  619-232-1234.
Awardee; The Irving Sigal Young Investigator Awardee;
The Stein and Moore Awardee; the Dorothy Crowfoot                        eMerGency MeDIcal servIces
Hodgkin Awardee; and the Presentation of the Eli Lilly and               In case of a medical emergency, contact the Symposium
Company Awards for Best Student Posters and recognition                  Management	Office,	Windsor	Room,	3rd	Level	or	dial	55	
of the Undergraduate Student Presentations.                              for hotel staff to assist you.
The award recognitions will be followed by the keynote
                                                                         eMPloyMenT oPPorTunITIes anD
address from invited speaker, Baldomero Olivera,
University of Utah, Distinguished Professor of Biology.
                                                                         recruITInG foruM
an exchange coupon has been given to each “full”                         The Protein Society has set aside a self-service Employment
member registrant. The coupon must be exchanged at                       Opportunities area in the Registration Area, Douglas Foyer.
the Ticket counter in the registration area for a ticket                 Attendees can post/review resumes and job openings in this
no later than 10:00 aM on Monday, august 2. Those who                    area. The Protein Society also maintains a free employment
purchase a ticket on-site will receive the actual ticket and             service for members and employers seeking opportunities.
do not need to make an exchange. Additional tickets may                  Visit	and	click	on	“Job	Opportunities.”	
be purchased at the Ticket Counter for $100 per person,                  The site includes searches for industrial, academic,
based upon availability. Please remember to bring the ticket             government and postdoctoral positions.
with you. You will not be admitted to the Awards Banquet
without a ticket. Complimentary drinks are limited to two                eXHIbITs
per person. If a vegetarian meal is desired, please inform               The Symposium exhibits are located in Douglas Pavilion
the Symposium Management Office, Windsor Room                            BCD on the 1st Level. A wide range of commercial exhibits
(3rd	Level)	by	10:00	am on Monday, August 2.                             of particular interest to protein scientists will be on display
                                                                         on Monday, 12:00 pm – 6:45 pm, Tuesday, 10:00 am – 6:45 pm
fred richards Memorial                                                   and Wednesday, 10:00 am – 3:30 pm.
A few tables will be reserved at the banquet for friends and
colleagues of Fred Richards. If you are interested in this               eXHIbIT ManaGeMenT offIce
gathering, you are welcome to take a seat at the marked                  The	management	office	is	located	in	the	Registration	Area,	
tables	at	the	banquet.	Seats	will	be	available	on	a	first-come-          Douglas	Foyer	on	the	1st	Level.		The	office	will	be	open:
first-served	basis;	prior	banquet	ticket	purchase	required.              Sunday, August 1 – 8:00 am – 5:00 pm
                                                                         Monday, August 2 – 10:30 am – 6:30 pm
baDGe/PublIcaTIons/ToTe baG                                              Tuesday, August 3 – 7:30 am – 6:30 pm
PIcKuP                                                                   Wednesday, August 4 – 7:30 am – 4:00 pm
Douglas Pavilion foyer, 1st level
                                                                         eXHIbITor WorKsHoPs
U.S. attendees who registered prior to July 1 received
their badge in advance of the meeting. All other registrants             Exhibitor Workshops will be presented Monday-Wednesday.
including	International	registrants	must	go	to	the	“Badge	               Please check the exhibitor workshops on pages 62 – 64 for
Pick-up” counter in the registration area. all attendees are             exact time and location.
required to wear their badge at all times. In addition to it

GraDuaTe ProGraM faIr                                                 MeMbers busIness MeeTInG
Manchester foyer, 2nd level                                           All members are encouraged and invited to attend the annual
The Mentoring Committee and the Education Committee                   Members Business Meeting on Tuesday, August 3 at 7:30
                                                                      am in Douglas Pavilion A on 1st Level. The advice and
of The Protein Society are proud to host the 4th Annual
Graduate Program Fair on Wednesday, August 4 from 12:15               guidance of the membership on current society issues and
pm to 1:45 pm. The fair is designed for students interested
                                                                      the	journal	are	welcome	in	this	“open	forum”	meeting.
in	 pursuing	 careers	 in	 the	 field	 of	 protein	 science.	 Each	
registered program will have materials and information                MeMbersHIP booTH
available to hand out to interested students.                         The Protein Society is the leading international society
                                                                      devoted to furthering research and development in
Green MeeTInG InITIaTIves                                             protein science. The purpose of The Protein Society is to
The Protein Society has implemented environmentally                   provide international forums to facilitate communication,
friendly initiatives for the 24th Annual Symposium                    cooperation and collaboration with respect to all aspects of
in San Diego. The Symposium tote bags are made of                     the study of proteins. In support of these goals, the society
polypropylene;	 partially	 recycled	 (from	 plastic)	 and	            also publishes Protein Science, the premier journal in the
100% recyclable material. The Symposium program, call                 field.	Members	have	an	opportunity	to	actively	participate	in	
for abstracts, exhibitor prospectus, and other materials are          the	emerging	fields	of	protein	science,	such	as	proteomics,	
printed on recycled paper. Plastic badge holders can be               bioinformatics, structural biology and computational
dropped in the collection bin at the registration desk. The           biology as they pertain to proteins at the molecular and
hotel selected for the Symposium is one of the leading                cellular level. The Protein Society members represent
companies in the hospitality management industry for                  academia,	industry,	government	and	non-profit	institutions	
promoting green initiatives. Manchester Grand Hyatt has               from around the world. Stop by The Protein Society booth
a Green Team and introduced a Green Meeting Initiative                to discuss your membership, ideas or suggestions with
in 2008. The following initiatives are in place at the                the Executive Officer, Cindy Yablonski, cyablonski@
Manchester Grand Hyatt: televisions, computer monitors,     
copiers, printers and other electronic devices are recycled;
recycling	containers	in	every	office	and	area	of	the	hotel;	          MessaGes
recycling program and education for employees to learn                Messages may be posted for attendees on the Message Board
how	to	help	at	work	as	well	as	at	home	installed	low-flow	            located in the Registration Area, Douglas Foyer. Please
showerheads and toilets; water/energy saving linen and                check for messages daily. Messages that are not picked up
towel program; meeting room recycling, where available;               by 11:00 am on Thursday, August 5 will be discarded.
online event menus; and paperless billing. The Society will
look for other green options and will continue to improve             neW MeMbers WelcoMe breaKfasT
its practices for future meetings.                                    All new members of The Protein Society are invited to
                                                                      attend the New Members Welcome Breakfast on Monday,
InTerneT/WIfI                                                         August 2 from 7:30 am to 8:30 am in Manchester D on the
All guest rooms at the Manchester Grand Hyatt have                    2nd Level. Coffee and light refreshments will be offered.
complimentary Internet access. Internet service is also
available at the Business Center on the 1st Level for a fee.          PosTer sessIons
WIFI access is available in the lobby for $9.95/day.                  Posters will be displayed in the Exhibit Hall/Douglas
                                                                      Pavilion, on the 1st Level from Monday through Wednesday.
losT anD founD                                                        A different set of posters will be displayed and presented
The Lost and Found is located in the Symposium                        each day. Presentation times for authors are listed at the
Management	Office,	Windsor	Room	on	the	3rd	Level.                     beginning of each poster session (refer to pages 29, 38,
                                                                      and	47). The poster board number is the alpha/numerical
Meal funcTIons                                                        listing	 (e.g.,	 B1)	 next	 to	 the	 abstract	 number.	Abstracts	
Meals are not included with the registration fee. The                 submitted after April 26 are printed in the Symposium
Awards Banquet is included in the Member registration                 Program Addendum.
fee only (it is not included in the student/postdoctoral              Poster Mounting schedule
registration).	Postdoctoral	fellows,	students	and	exhibitors	
                                                                      Monday, Poster session 1 presenters must place poster material
may purchase tickets for the banquet based on availability
                                                                      on assigned board between the hours of 7:00 am – 8:00 am
at the Registration Desk in the Douglas Foyer by 10:00 am
                                                                      and remove posters the same evening between the hours
on Monday, August 2.
                                                                      of 6:45 pm – 7:15 pm.

Tuesday, Poster session 2 presenters must place poster material          TelePHone nuMbers
on assigned board between the hours of 7:00 am – 8:00 am                 Symposium/Exhibit Management                 619-358-6826
and remove posters the same evening between the hours of                 Registration Desk                            619-358-6871
6:45 pm – 7:15 pm.                                                       AV	Office	                                   619-358-6827
Wednesday, Poster session 3 presenters must place poster                 Manchester Grand Hyatt                       619-232-1234
material on assigned board between the hours of 7:00 am –                Emergency/First Aid: dial 55 from any house phone in the
8:00 am and remove material the same afternoon between                   hotel
the hours of 3:15 pm – 3:30 pm.                                          Security: dial 4203 from any house phone in the hotel
Please do not leave belongings, poster containers, or any
materials under the poster boards or in the poster area.                 TourIsT InforMaTIon
The Protein Society is not responsible for articles left in              Information regarding tours will be available at the
the poster area.                                                         Concierge desk on the 1st Level of the Manchester Grand
                                                                         Hyatt. For additional information on the sites and sounds of
Please refer to the symposium Program addendum for                       San Diego, visit the city’s website at
full listing of late-breaking addendum abstracts and
updated floor Plan with poster board numbers.                            TransPorTaTIon
                                                                         San Diego is served by the San Diego International Airport.
ProGraM/absTracT booK                                                    The airport is located about three miles/10 minutes from
Each registrant will receive one copy of the Symposium                   the Manchester Grand Hyatt and downtown San Diego. For
Program Abstract book. Additional copies may be purchased                further information visit
in the Registration Area, Douglas Foyer for $15 per book.
                                                                         shared van service
scIenTIfIc sessIons                                                      Shared van service to and from San Diego International
The sessions begin Sunday, August 1 and end Wednesday,                   Airport is generally available every day from early
August 4. Please consult the daily schedule for further                  morning until well after midnight. Contact the individual
details.                                                                 operators for schedules, fares, and reservations.
                                                                         The following companies provide service between San Diego
sMoKInG PolIcy                                                           International Airport and the greater San Diego area:
Smoking is not permitted in the hotel.
                                                                         access shuttle
                                                                         800-690-9090, 619-282-1515
Presenters are asked to arrive in the session room at least
a 1/2 hour prior to the start time of the session to set up              advanced shuttle
their laptop and check in with the projectionist. If you                 800-719-3499
would like to practice your presentation prior to the day of   
presentation,	please	let	the	technicians	know	in	AV	Office	              airport shuttle
located	in	the	Oxford	Room	on	the	3rd	Level.	The	office	                 888-254-0333, 619-234-4403
is open 4:30 pm – 8:30 pm Saturday, 7:30 am – 5:00 pm                    Cloud 9 Shuttle/Super Shuttle
Sunday – Wednesday and 7:30 am – 12:00 pm on Thursday.                   800-9-SHUTTLE	(974-8885)
Data projectors will be provided in all session rooms.         
Presenters must bring their own laptops to the meeting.
                                                                         eZ ride
sPecIal neeDs
For on-site assistance, please visit the Symposium
Management	Office,	Windsor	Room	on	the	3rd	Level.                        Prime Time shuttle
                                                                         800-REDVANS	(733-8267)
syMPosIuM ManaGeMenT offIce                                    
The	management	office	is	located	in	the	Windsor	Room	on	                 sea breeze shuttle
the	3rd	Level.		The	office	will	be	open:                                 619-297-7463
Saturday, July 31 – 4:30 pm – 8:30 pm                          
Sunday, August 1 – Wednesday, August 4 – 7:30 am – 5:00 pm
Thursday, August 5 – 8:00 am – 12:00 pm

Taxis                                                                between Terminals 1 and 2 and the Commuter Terminal.
Many companies provide taxicab service at San Diego                  The 10-minute bus service connects with Trolley, Coaster
International Airport. If you need a taxi, simply follow the         and Amtrak stations and is wheelchair accessible. Planning
signs leading to the Transportation Plazas. A Transportation         a trip is easy when you use the Online Transit Information
Coordinator	will	place	you	with	the	first	available	taxi	unless	     System. For more information, call 619-699-1900.
you specify a particular taxicab company.
Public Transit                                                       The San Diego trolley does not stop at the airport. Refer to
Public transit is available to and from the Airport and              the trolley map to locate the routes and stops throughout
downtown San Diego on Flyer Route No. 992, stopping                  the city in addition to fares and time schedule.

                                                    cell/Mobile Phones
                As a courtesy to your fellow attendees, please turn off all cell phones and beepers prior to
                                                 entering a session room.

                                                cameras/video recording
                          The use of cameras/video recording inside session rooms is prohibited.

                                     syMPosIuM HIGHlIGHTs

behind the nIH study section Door: nIH                               structural biology, molecular and computational biology,
Mock review Panel                                                    and biophysics—will have the opportunity to interact with
Monday, August 2, 12:15 pm – 1:15 pm                                 representatives from institutions with graduate programs
Manchester D, 2nd Level                                              that have registered to participate in the fair.

This workshop will provide an introduction into the review              !
process of an NIH research grant application. Experienced
investigators as well as representatives from NIH will               How to find a Postdoc Position
present a mock review panel – providing an accurate                  Tuesday, August 3, 7:30 pm – 9:00 pm
glimpse at the internal review process.                              Manchester D, 2nd Level
                                                                     Are your e-mails inquiring about postdoc positions going
career Panel                                                         unanswered? In this workshop, we will provide some
Tuesday, August 3, 12:00 pm – 1:00 pm                                “tricks	of	the	trade”	to	best	present	your	application	for	a	
Manchester G, 2nd Level                                              postdoctoral position in a top laboratory.
In this session, young protein scientists can shape their
careers and the future workforce. The panel will help young     NEW
scientists make decisions on pathways that may lead to               How to Give a Great Talk
industrial or academic careers. Sponsor representatives may
                                                                     Monday, August 2, 7:00 pm – 8:30 pm
participate in the panel discussions.
                                                                     Manchester D, 2nd Level
                                                                     Ever sat through a boring talk and vowed never to give one?
educator’s luncheon Workshop: exploring
                                                                     In this workshop, we will provide clear guidelines on the
the rcsb Protein Data bank                                           do’s and dont’s of giving a great talk.
(ticket purchase required)
Sunday, August 1, 12:15 pm – 1:45 pm                                 Mentoring comittee and education committee
Elizabeth F, 2nd Level                                               Meeting & Mixers
The Educators’ luncheon is a topic-oriented workshop                 Sunday, August 1, 7:30 pm – 9:00 pm
aimed at assisting faculty in keeping their courses current          Gallery Room – Exit Seaport Tower by Lae’s Restaurant,
in light of rapid developments in protein science.                   Lobby Level. It is located in the Seaport Courtyard, next
                                                                     to Ann Marie’s Coffee Shop.
Fundamentals of Good Scientific Writing                              This mixer is the primary opportunity for young protein
(special registration required)                                      scientists	(students,	postdocs	and	young	faculty)	to	become	
Wednesday, August 4, 12:30 pm – 1:30 pm                              acquainted with each other. It’s a chance to interact with
Manchester G, 2nd Level                                              career building individuals. Mixer sponsors are also seeking
Can’t find the right word or phrasing for your latest                to engage participants in committee activities.
paper? In this workshop, we will provide useful tools
for	clear	scientific	writing	and	demonstrate	these	tools	by	         networking Dinners
deconstructing and reconstructing an abstract solicited from         Monday, August 2, 7:15 pm; Tuesday, August 3, 7:15 pm
participants.                                                        Postdocs and students can network with distinguished
                                                                     Symposium colleagues—Speakers, Councilors, Award
Graduate Program fair                                                Winners—at selected area restaurants. Several small dinner
Wednesday, August 4, 12:15 pm – 1:45 pm                              groups will be arranged to include about 10 participants
Manchester Foyer, 2nd Level                                          each	at	area	restaurants.	First	come,	first	served;	self	pay.	
The Mentoring Committee will host the Fourth Annual                  Sign up in the registration area in the Douglas Foyer, 1st
Graduate Program Fair at the Symposium. Students                     Level. The list of dinner leaders will be included in the
interested in pursuing graduate training and careers in              Symposium Program Addendum.
the field of protein science—including biochemistry,

resume Writing luncheon Workshop                             student/Postdoc lounge
(ticket purchase required)                                   Douglas Pavilion/Exhibit Hall, 1st Floor
Sunday, August 1, 12:15 pm – 1:45 pm                         Undergraduate and graduate students and postdocs can
Elizabeth G, 2nd Level                                       network with each other and with mentor and career
Does your resume best reflect your skills and                associates or just regroup and relax in this lounge.
accomplishments? Hiring experts in academia and industry
will offer practical advice on how to develop an effective   undergraduate student research session
resume and hone your job hunting and interviewing skills.
                                                             Wednesday, August 4, 1:30 pm – 3:10 pm
These workshops are designed to ensure that you are
                                                             Manchester ABC, 2nd Level
marketing your skills to the best of your ability.
                                                             Undergraduate students will present their work to gain
                                                             visibility and make connections for graduate school and for
                                                             professional positions. Be sure to attend this session!

      vIsIT THe eXHIbIT Hall
              See a wide range of commercial exhibits of particular
                          interest to protein scientists!

                          exhibit Hall – Douglas Pavilion bcD
                                           12:00 pm – 6:45 pm
                                           10:00 am – 6:45 pm
                                         10:00 aM – 3:30PM

    Refer to pages 57 – 61 for detailed information on exhibitor products.

                         fuTure MeeTInG DaTes

25th annual symposium        27th annual symposium    29th annual symposium
   July 23 – 27, 2011           July 20 – 24, 2013       July 25 – 29, 2015
 boston, Massachusetts        boston, Massachusetts    boston, Massachusetts
26th annual symposium        28th annual symposium    30th annual symposium
   august 5 – 9, 2012           July 26 – 30, 2014       July 9 – 13, 2016
 san Diego, california        san Diego, california    san Diego, california


    2O1    1AWARDS                                                                                              2O11
    of The Protein Society
The Protein Society is seeking
                                                        NOMINATIONS ACCEPTED
nominations for its 2011 awards.                        FOR THE FOLLOWING AWARDS:
The Brändén and Hodgkin awards will be                  Carl Brändén Award: In the tradition of the late Carl Brändén,
presented at the IX European Symposium of               pioneer in structural biology, co-author of the seminal text,
The Protein Society in Stockholm, Sweden,               Introduction to Protein Structure, and leader of the world-class
May 22–26, 2011.                                        synchrotron facility at Grenoble, the Carl Brändén Award, sponsored
                                                        by Rigaku Corporation, is given to an outstanding protein scientist
The Neurath, Anfinsen, Stein and Moore, Kaiser,
                                                        who has also made exceptional contributions in the areas of education
and Sigal awards will be presented at The 25th          and/or service to the science.
Annual Symposium of The Protein Society in
Boston, Massachusetts, July 23–27, 2011.                Dorothy Crowfoot Hodgkin Award: The Dorothy Crowfoot
                                                        Hodgkin Award, sponsored by Genentech, is granted in recognition of
Recipients will present plenary lectures at the         exceptional contributions in protein science, which profoundly
symposia on the structure and function of protein       influence our understanding of biology.
science as it relates to their field of study.
                                                        Stein and Moore Award: The Stein and Moore Award,
Deadline: Complete award nominations                    sponsored by The Protein Society and named for Nobel Laureates Dr.
packages for the 2011 Awards cycle must be              William Stein and Dr. Stanford Moore, venerates their contribution to
                                                        understanding the connection between chemical structure and
submitted by October 1, 2010. The Protein
                                                        catalytic activity of the active center of the ribonuclease molecule. To
Society requires that all nominees be living at the
                                                        that end, recipients of the Stein and Moore award are recognized for
time of an award nomination.                            their contributions to the highest level of the study of proteins.

How to nominate:                                        Hans Neurath Award: The Hans Neurath Award, sponsored by
                                                        the Hans Neurath Foundation, recognizes an individual who has made
Nominations should be e-mailed by the October           a recent contribution of unusual merit to basic research in the field of
1, 2010 deadline to:          protein science, including but not restricted to the chemistry, design,
                                                        folding, structure, or biological function or proteins.
Please note that the Society will only accept
submissions in PDF format—preferably one PDF            Christian B. Anfinsen Award: The Christian B. Anfinsen
per single, complete award nomination package.          Award, sponsored by The Aviv Family Foundation, recognizes
                                                        significant technical achievements in the field of protein science.
For more information, please visit the 2011
Nominations page on the Web at:                         Emil Thomas Kaiser Award: The Emil Thomas Kaiser Award,
                                                        sponsored by The Protein Society, recognizes a recent, highly
                                                        significant contribution in applying chemistry to the study of proteins.
Also note that previous nominations are
                                                        Irving Sigal Young Investigator Award: The Irving Sigal
automatically carried over for two years;               Young Investigator Award, sponsored by Merck Research Laboratories,
subsequent renominations must be submitted as           is an award that recognizes an important contribution to the study of
indicated above.                                        proteins by a scientist who is in the early stages of an independent
                                                        career. Candidates are generally not more than 40 years of age.
We look forward to receiving your nominations.

                   Questions? Please contact Cindy A. Yablonski, Executive Officer, The Protein Society
                • (301) 634-7277 or (800) 99-AMINO
                                                                                                floor Plan

     FOURTH LEVEL                                                                                     AMERICA’S CUP
                                                                                                                             WHIRL POOL           FIRE PITS                          SEAPORT VILLAGE
                                                                               CUNNINGHAM                                                                      WHIRL POOL

                                                                                                      A             A                                                                                    FITNESS CENTER

                                                                                          FOYER B                    B

                                                                        ESCALATORS ELEVATORS
                                                                                                          C             C

                                             RANDLE TERRACE                                                                 D                                                                                                            RESTROOMS

                                             RANDLE FOYER
                                                                                                                     RESTROOMS                                 STAGE

                                                  D     C    A                                  GIBBONS
                                                                                                                          AMERICA’S                                                         ELEVATORS
                                                      RANDLE                       RESTROOMS
                                                                                                               AMERICA’S CUP TERRACE                                  SEASONAL
                                                     BALLROOM                                                                                                      POOL BAR & GRILLE
                                                                                                               CUP FOYER
                                                   E          B                          ROOF-TOP
                                                                                       SPORT COURTS


     THIRD LEVEL                                               POOL TERRACE                                                     C     B        A       B       A               ESCALATORS

                                                                    SPA POOL
                                                                                            FOYER                                         EMMA ANNIE MAGGIE                                    R
                                                  WHIRL POOL                                                                                                                                       ES
                                                                                               ELEVATORS                                                                                                         O
                                                                        ESCALATORS                                                                                                                                   M

                                                                                                                    OXFORD                                                  FREIGHT
                                                                       KIN SPA                                                                                             ELEVATORS                                                     GEORGE BUSH
                                                                                                                                                                                                                                 A            FORD
                                                                               RESTROOMS                  CONNAUGHT                                                                                                                  B
                                                                                                                            SHOW MANAGER
                                                                                                                               OFFICE 3                                                                                                  C
                                                                                                              SHOW MANAGER                            ELIZABETH TERRACE
                                                      MANCHESTER                                                 OFFICE 2                                                                    MOHSEN                                                       MADELEINE
                                                                                                                                                                                                                             A               A
     SECOND LEVEL                                                           SHOW MANAGER
                                                                               OFFICE 5                                                                                                                                      B                    B

                                                                                                               LITRENTA                       ELIZABETH BALLROOM                                                     DEL             A                C
                              SHOW MANAGER                                              PALM                    FOYER                                                                                                MAR
                                                                                                                                                                                                                                     B                D

                                 OFFICE 1                                              COURT

                                                                                                                                      F                                          A

                                                                                                ELEVATORS                                                                                                                                                          PSAV OFFICE

                                                                                         RESTROOMS       RESTROOMS

                                                                                                                                                           E        D
 TOWER                                                                                                                                    G                                          B                                                           RESTROOMS                        SEAPORT
                                         G                  D                  A                                                                   ELIZABETH FOYER                                                                                                                TOWER
                                               MANCHESTER                                                                                                                                                                            A
                                                                                                                                              H                                      C                                                           BETSY
                                                BALLROOM                                                                                                                                                                                          ELEVATORS
                                             H      E                          B                 ESCALATORS

                                                                                                                                                                             FREIGHT                                                      C
                                              I                 F                  C                                 SHOW MANAGER
                                                                                                                        OFFICE 4
                                                                                                                                                                                                                             A                A           EDWARD
                                                                                                                                          PAVILION LOAD-IN
                                                                                                                                                                                                                                 B                B
                                                                                                                                                                                                   GREGORY                           A                C

                                                                                                                                                                                                                                          B               D
                                                                                   RESTROOMS                                                                                                                      FREIGHT
                                         LAEL’S RESTAURANT                                                                                                                                DOCK                   ELEVATORS

                                                                                                                            D                 C                B                A

                                                                       PROMENADE                                                          DOUGLAS PAVILION
                                                            TOP OF THE HYATT                                                                                                                                                              ESCALATORS
                                                      (ELEVATOR TO THE 40TH FLOOR)
                                                                                                                                                   DOUGLAS FOYER                                                                                      ELEVATORS

                                                                             GRAND                                            DESK                             FRONT DESK            BUSINESS CENTER
                                                                           LOBBY BAR

                                                  CONVENTION                                                                                                                                                                                                  REDFIELD’S DELI
                                                    CENTER                                                                          CAR RENTAL                          BELL
                                                                         DOWNTOWN AND                                                  DESK                             DESK
                                                                        GASLAMP DISTRICT                                                                            MAIN ENTRANCE                                                                                    REDFIELD’S
                                                                                                                                                                                                                                                                    SPORT’S BAR


                Sunday, August 1                           Monday, August 2                             Tuesday, August 3                       Wednesday. August 4                     Thursday, August 5
                                                                                                                            st                                      st                                    st
           Douglas Pavilion BCD, 1st Level          Douglas Pavilion BCD, 1st Level              Douglas Pavilion BCD, 1 Level             Douglas Pavilion BCD, 1 Level           Douglas Pavilion BCD, 1 Level
               12:00 PM – 5:00 PM                         7:00 AM – 8:00 AM                             7:00 AM – 8:00 AM                         7:00 AM – 8:00 AM                      8:00 AM – 2:00 PM
                Exhibit Installation                          Posters set up                               Posters set up                            Posters set up                           Move-out
                                                           8:00 AM – 6:45 PM                            8:00 AM – 6:45 PM                         8:00AM – 3:15 PM
                                                        Posters Open for Viewing                     Posters Open for Viewing                  Posters Open for Viewing
                                                            12 PM – 6:45 PM                             10:00 AM – 6:45 PM                       10:00AM – 3:30 PM
                                                              Exhibits Open                                 Exhibits Open                            Exhibits Open
                                                           1:15 PM – 2:45 PM                           10:00 AM – 10:45 AM                      10:00 AM – 10:45 AM
                                                  Poster Session 1; poster judging (half)                   Refreshments                             Refreshments
                                                           2:00 PM – 2:45 PM                             1:15 PM – 2:45 PM                        1:45 PM – 3:15 PM
                                                              Refreshments                     Poster Session 2; poster judging (half)             Poster Session 3
                                                           5:30 PM – 6:45 PM                             2:00 PM – 2:45 PM                        2:00 PM – 2:45 PM
                                                             Munch N Mingle                                 Refreshments                             Refreshments
                                                           6:45 PM – 7:15 PM                             5:30 PM – 6:45 PM                Posters must be removed between
                                                            Posters Removal                                Munch N Mingle                         3:15 PM – 3:30 PM
                                                                                                         6:45 PM – 7:15 PM
                                                                                                          Posters Removal                    3:30 PM – Exhibitor Move-out
               Elizabeth DE, 2nd Level                 Manchester ABC, 2nd Level                    Manchester ABC, 2nd Level                Manchester ABC, 2nd Level               Manchester ABC, 2nd Level
               9:00 AM – 12:00 PM                        9:00 AM – 12:00 PM                              2:45 PM – 5:20 PM                       9:00 AM – 12:15 PM                     9:00 AM – 12:00 PM
             Symposia 2: Imaging and                Symposia 6: Disordered Proteins                Symposia 10: Metalloproteins            Symposia 12: Amyloids & Disease               Plenary Session 2:
          Deconstructing Large Complexes                  2:45 PM – 5:20 PM                                                                       1:30 PM – 3:10 PM                   Macromolecular Machines
                2:00 PM – 4:45 PM                     Symposia 7: Protein Folding                                                          Undergraduate Student Research
       Symposia 4:Visualizing Proteins in Cells                                                                                                        Session
                5:00 PM – 7:10 PM                                                                                                                 3:20 PM – 6:20 PM
             Awards Plenary Session 1                                                                                                          Awards Plenary Session 2
            Douglas Pavilion A, 1st Level             Douglas Pavilion A, 1st Level                 Douglas Pavilion A, 1st Level           Douglas Pavilion A, 1st Level
               9:00 AM – 12:00 PM                         9:00 AM – 12:00 PM                             7:30 AM – 8:30AM                        9:00AM – 12:15 PM
        Symposia 1: Protein Engineering for       Symposia 5: Protein Therapeutics and               Member Business Meeting             Symposia 11:Protein Design &Evolution
            Solubility and Reconstitution                      Diagnostics                              9:00AM – 11:45 AM
                2:00 PM – 4:45 PM                          2:45PM – 5:20 PM                  Plenary Session 1: Fred Richards Memorial

       Symposia 3: Protein Stability and Drug      Symposia 8: Membrane Proteins &                       2:45 PM – 5:20 PM
                     Formulation                               Receptors                     Symposia 9: Microfluidics and Microarrays
                Elizabeth F, 2nd Level                   Manchester D, 2nd Level                      Manchester D, 2nd Level                  Manchester D, 2nd Level
               12:15 PM – 1:45 PM                          7:30 AM – 8:30AM                             12:00 PM – 1:00 PM                       12:30 PM – 1:30 PM
           Educators Luncheon Workshop              New Member Welcome Breakfast                Exhibitor Workshop: Sierra Sensors             Exhibitor Workshop: TCxT
         (advance ticket purchase required)               12:15 PM – 1:15 PM                             7:30 PM – 9:00 PM
                                                   Behind the NIH Study Section Door:             How to Find a Postdoc Position
                                                         A Mock Review Panel
                                                           7:30 PM – 9:00 PM
                                                        How to Give a Great Talk
               Elizabeth G, 2nd Level                   Manchester E, 2nd Level                        Manchester E, 2nd Level                Manchester Foyer, 2nd Level
               12:15 PM – 1:45 PM                         12:15 PM – 1:15 PM                             12:00 PM – 1:00 PM                      12:15 PM – 1:45 PM
         Resume Writing Luncheon Workshop            Exhibitor Workshop: ForteBio              Exhibitor Workshop: Olink Bioscience             Graduate Program Fair
          (advance ticket purchase required)
         Gallery Room, Seaport Courtyard                                                               Manchester F, 2nd Level                 Manchester F, 2nd Level
                7:30PM – 9:00 PM                                                                        12:00 PM – 1:00 PM                        12:30 PM – 1:30 PM
           Mentoring/Education Committee                                                      Exhibitor Workshop : Wyatt Technology       Exhibitor Workshop : GE Healthcare
                  Meeting & Mixers
                                                                                                      Manchester G, 2nd Level                 Manchester G, 2nd Level
                                                                                                        12:00 PM – 1:00 PM                      12:30 PM – 1:30 PM
                                                                                                           Career Panel                  Fundamentals of Good Scientific Writing
                                                                                                       Manchester H, 2nd Level               Manchester ABC, 2nd Level
                                                                                                        12:00 PM – 1:00 PM                       Awards Banquet
                                                                                                Exhibitor Workshop : GE Healthcare           Reception at 7:30 PM Foyer
                                                                                                                                                Banquet at 8:00 PM
                                                                                                                                           Advance ticket purchase required
                                                   Check sign up board in the Douglas           Check the sign up board in the Douglas
                                                              Foyer – 1st Level                              Foyer – 1st Level
                                                  7:15 PM– 9:30 PM Networking Dinners             7:15 PM– 9:30 PM Networking Dinners
                                                        (self pay - sign up onsite)                     (self pay - sign up on site)
                                                                                Please refer to the Symposium Addendum for changes/updates.
                    SATURDAY, JULY 31

                         6:00 PM – 8:30 PM
                   Douglas Pavilion Foyer, 1st Level

                    SUNDAY, AUGUST 1

                         7:30 AM – 6:00 PM
                   Douglas Pavilion Foyer, 1st Level

                         8:00 AM – 5:00 PM
                   Douglas Pavilion Foyer, 1st Level

                    NETWORKING DINNERS
                         MONDAY, AUGUST 2
                         TUESDAY, AUGUST 3
                              7:15 PM

     Sign up on-site in the Registration Area, Douglas Foyer 1st Level.
                     (First-come, first-served, self-pay.)

Dinner leaders are listed in the Symposium Program Addendum.

                                     SUNDAY, AUGUST 1
                          CONCURRENT MORNING SYMPOSIA 1
                               9:00 AM—12:00 PM
                            Douglas Pavilion A, 1st Level

Session Chair: Karyn O’Neil, Johnson & Johnson Internal Ventures
           Abstract #
9:00 AM                 Protein Tagging and Labeling Systems for Protein Chemistry and
                        Cellular Biology
                        G. Waldo. Los Alamos Natl. Lab.
9:30 AM        1        Electrostatic Effects in Proteins: Charges and Consequences
                        J.M. Scholtz. Texas A&M Hlth. Sci. Ctr. Col. of Med.
10:00 AM                Break (refreshments outside room)
10:30 AM                TBD
                        D. Minor. UCSF.
11:00 AM       315      Young Investigator Talk
                        A facile Preparation of Ubiquitinated Proteins Using Pyrrolysine
                        Analogs X. Li. The Ohio State Univ.
11:15 AM       159      Young Investigator Talk
                        Antibody fragment Engineering with Non-canonical Amino Acids
                        J. Van Deventer. Caltech.
11:30 AM       2        Improving Protein Solubility: An Evolutionary Approach Using
                        E.coli DHfR as a Reporter
                        D. Ollis. Australian Natl. Univ.

                                     SUNDAY, AUGUST 1
                          CONCURRENT MORNING SYMPOSIA 2
                                    9:00 AM—12:00 PM
                                   Elizabeth DE, 2nd Level

Session Chair: Wolfgang Baumeister, Max Planck Inst. for Biochem.
           Abstract #
9:00 AM        3        DNA Repair Machines, Disease Causing Mutations, and Insights
                        for Cancer Interventions
                        J. Tainer. The Scripps Res. Inst.
9:30 AM        4        Dicer and Beyond: Making and Using RNA for Genetic Control
                        J. Doudna. Univ. of California, Berkeley/HHMI.
                                                                                          J. Tainer
10:00 AM                Break (refreshments outside room)
10:30 AM       5        A Hybrid Approach to Understanding Virus Structure and
                        S. Butcher. Univ. of Helsinki.
11:00 AM       198      Young Investigator Talk
                        Contact Surface Mapping of LysRS in Its Multi-functional States
                        by Solution-Phase Hydrogen/Deuterium Exchange fT-ICR Mass         J. Doudna
                        H-M. Zhang. Natl. High Magnetic Field Lab., Florida State Univ.
11:15 AM       298      Young Investigator Talk
                        Mechanisms and Atomic Structures of the Shell Proteins from a
                        Bacterial Organelle in Salmonella enterica
                        C. Crowley. UCLA.
11:30 AM       6        The Organisation and Dynamics of Molecular Chaperone              S. Butcher
                        J. Buchner. Tech Univ. Munich.

                                                                                          J. Buchner

                   SUNDAY, AUGUST 1
                CONCURRENT WORKSHOP 1
                    12:15 PM—1:45 PM
                   Elizabeth F, 2nd Level
            Chair: David Bevan, Virginia Polytechnic Univ.
           (advance registration and ticket purchase required)
                   D. Goodsell, The Scripps Res. Inst.

                CONCURRENT WORKSHOP 2
                    12:15 PM—1:45 PM
                   Elizabeth G, 2nd Level
Hiring experts offer practical advice on developing an effective resume
          and improving job search and interviewing skills.
                   (advance ticket purchase required)
                 Chair: Blake Hill, Johns Hopkins Univ.

                                     SUNDAY, AUGUST 1
                        CONCURRENT AfTERNOON SYMPOSIA 3
                                      2:00 PM—4:45 PM
                                  Douglas Pavilion A, 1st Level

Session Chair: John Carpenter, Univ. of Colorado Denver.
Session organized by John Carpenter, Univ. of Colorado, Denver and Ted Randolph, Univ. of Colorado at Boulder.
           Abstract #
2:00 PM        7        Trisulfide Modification: Impact on Monoclonal Antibody Stability
                        K. Lucas. Biogen Idec, San Diego.
2:30 PM        8        New and Evolving Analytical Methods for Evaluating Protein
                        A. Herman. Althea Technologies Inc., Camarillo, CA.
3:00 PM        325      Young Investigator Talk
                        Engineering High Affinity Knottin Peptides Targeting Tumor
                        Marker CAIX for Cancer Imaging and Therapy
                        S. Moore. Stanford Univ.
3:15 PM                 Break (refreshments outside room)
3:45 PM        9        Agitation-Induced Aggregation of Monoclonal Antibodies and the
                        Role of Container Closure Systems
                        T. Estey. Biogen Idec, San Diego.
4:15 PM        10       Improving Physicochemical Properties of Biopharmaceutical Drug
                        D. Katatyama. Amylin Pharmaceuticals, San Diego.

                                     SUNDAY, AUGUST 1
                        CONCURRENT AfTERNOON SYMPOSIA 4
                                 VISUALIzING PROTEINS IN CELLS
                                         2:00 PM—4:45 PM
                                       Elizabeth DE, 2nd Level

Session Chair: Ineke Braakman, Utrecht Univ.
           Abstract #
2:00 PM                 Dynamics of Membrane Proteins in Domains with fluid Attributes
                        Regulating Glucose Transport and Endocytosis
                        J. Zimmerberg. NICHD, NIH.
2:30 PM        11       Protein folding in the ER: Life and Death Decisions
                        L. Hendershot. St. Jude Children’s Res. Hosp.
3:00 PM        269      Young Investigator Talk
                        Imaging of Mobile Stable Lipid-Mediated Protein Associations in
                        the Live Cell Plasma Membrane
                        M. Brameshuber. Univ. of Linz, Austria.                            L. Hendershot

3:15 PM                 Break (refreshments outside room)
3:45 PM        12       NMR Observation of Structure, function and Stability of Proteins
                        inside Mammalian Cells
                        M. Shirakawa. Grad. Sch. of Engin., Kyoto Univ.
4:15 PM        13       Putting Molecules in Context with Correlated fluorescence and
                        X-ray Tomography
                        C. Larabell. UCSF.

                                     SUNDAY, AUGUST 1
                                 AWARDS PLENARY SESSION 1
                                            5:00 PM—7:10 PM
                                        Douglas Pavilion A, 1st Level

Session Chair: TBD
           Abstract #
5:00 PM                 Opening Remarks
5:05 PM                 The Carl Brändén Award (Sponsored by Rigaku Corporation)
                        Presenter: Paul Swepston, Sr. Advisor to the CEO of Rigaku
5:10 PM       14        Seeking General Views to Look at Proteins
                                                                                                    N. Go
                        N. Go. RIKEN Harima Inst., Hyogo, Japan.
5:40 PM                 The Christian B. Anfinsen Award
                        (sponsored by The Aviv Family Foundation Corporation)
                        Presenter: Jack Aviv (The Aviv Family Foundation)
5:45 PM       15        Structural Physiology Based on Electron Crystallography
                        Y. Fujiyoshi. Grad. Sch. of Sci., Kyoto Univ.
6:15 PM                 Break                                                                     Y. Fujiyoshi

6:30 PM                 The Emil Thomas Kaiser Award (sponsored by The Protein Society)
                        Presenter: Daniel Raleigh (Suny at Stony Brook), President, The Protein
6:35 PM       16        Structure, function, and Inhibition of Human O-GlcNAc
                        S. Walker. Harvard Med. Sch.
                                                                                                   S. Walker
7:05 PM                 Closing Remarks

                                      MEETING & MIXER
                                        7:30 PM – 9:00 PM
                                 Gallery Room, Seaport Courtyard
                           All registered attendees are invited to attend!
                           Cash bar and complimentary snacks will be available.
                                  Badges must be worn for admittance.

       7:30 AM – 8:30 AM
      Manchester D, 2nd Level

        7:30 AM – 6:00 PM
      Douglas Foyer, 1st Level

            8:00 AM – 6:45 PM
  Exhibit Hall/Douglas Pavilion, 1st Level

                                    MONDAY, AUGUST 2
                          CONCURRENT MORNING SYMPOSIA 5
                                      9:00 AM—12:00 PM
                                   Douglas Pavilion A, 1st Level

Session Chair: Wayne Fairbrother, Genentech.
           Abstract #
9:00 AM        17       Stapled a-Helical Peptides: The Twists and Turns of Drug
                        T. Sawyer. Aileron Therapeutics Inc., Cambridge, MA.
9:30 AM        18       Two-in-One Antibody That Interacts with Two Different Molecules
                        at the Antigen-Binding Site with High Affinity                     T. Sawyer
                        G. Fuh. Genentech.
10:00 AM                Break (refreshments outside room)
10:30 AM       19       Engineering Ultra-Stable Molecular Recognition Proteins Using
                        the Top7 Protein Scaffold
                        C. Baird. Pacific Northwest Natl. Lab., Richland, WA.
11:00 AM       422      Young Investigator Talk
                                                                                           C. Baird
                        Recognition of Influenza Virus Hemagglutinin by Neutralizing
                        D. Ekiert. The Scripps Res. Inst.
11:15 AM       407      Young Investigator Talk
                        Selective Prodrug Activation in the Presence of the Tumor-Marker
                        HIf-1a by Engineered Protein Switches
                        C. Wright. Johns Hopkins Univ.
11:30 AM       20       Delivering Antibody Therapeutics to Where the Action Is—Inside
                        the Cell
                        A. Kossiakoff. Univ. of Chicago.

                                    MONDAY, AUGUST 2
                          CONCURRENT MORNING SYMPOSIA 6
                                        DISORDERED PROTEINS
                                           9:00 AM—12:00 PM
                                        Manchester ABC, 2nd Level

Session Chair: Jean Baum, Rutgers Univ.
           Abstract #
9:00 AM        21       Relating Primary Sequence to Protein function in Intrinsically
                        Disordered Proteins by NMR
                        M. Blackledge. Univ. Joseph Fourier, CNRS, IBS.
9:30 AM        22       Structural Reorganization of a-Synuclein with pH Observed by
                        Replica Exchange Molecular Dynamics Simulations and Nulear
                        Magnetic Resonance
                        R. Levy. Rutgers, The State Univ. of New Jersey, Piscataway.
10:00 AM                Break (refreshments outside room)
10:30 AM       23       Probing Interactions in Unfolded Proteins with Single Molecule
                        fluorescence Spectroscopy
                        B. Schuler. Univ. of Zurich.
11:00 AM       444      Young Investigator Talk
                        Amyloid formation Kinetics and fibrillar Structures of the
                        Essential Melanosomal Protein Pmel17
                        J. Lee. NHLBI, NIH.
11:15 AM       327      Young Investigator Talk
                        Effects of Intrinsic Disorder in Regulating the Balance between
                        Intra- and Inter-molecular Interactions in the Signaling Adapter
                        H. Cheng. Fox Chase Cancer Ctr.
11:30 AM       24       Structural Disorder and Aberrant Proteins in the Cell
                        P. Tompa. Inst. of Enzymol., Hungarian Acad. of Sci., Budapest.

                   MONDAY, AUGUST 2
                       10:30 AM – 6:30 PM
                     Douglas Foyer, 1st Level

                           EXHIBITS OPEN
                          12:00 PM – 6:45 PM
                 Exhibit Hall/Douglas Pavilion, 1st Level

                      EXHIBITOR WORKSHOP
                         12:15 PM – 1:15 PM
                       See page 62 for full listing.

                           WORKSHOP 3
                    12:15 PM—1:15 PM
                   Manchester D, 2nd Level
  Experienced investigators and NIH representatives stage the internal
             review process of an NIH grant application.
                 Chair: Blake Hill, Johns Hopkins Univ.

                         POSTER SESSION 1
                           1:15 PM – 2:45 PM
                 Exhibit Hall/Douglas Pavilion, 1st Level

                           2:00 PM – 2:45 PM
                 Exhibit Hall/Douglas Pavilion, 1st Level

                                    MONDAY, AUGUST 2
                        CONCURRENT AfTERNOON SYMPOSIA 7
                                         PROTEIN fOLDING
                                           2:45 PM—5:20 PM
                                        Manchester ABC, 2nd Level

Session Chair: Jane Clarke, Univ. of Cambridge.
           Abstract #
2:45 PM        25       Protein folding/Unfolding Under force
                        J. Fernandez. Columbia Univ.
3:15 PM        26       Proteins Under Tension: Insights from Single-Molecule force
                        G. Hummer. NIDDK, NIH.
3:45 PM        173      Young Investigator Talk
                        Physical-Chemical Determinants of Coil Conformations in
                        Globular Proteins
                        L. Perskie. Johns Hopkins Univ.
4:00 PM                 Break
4:20 PM        27       A Tangled Problem: The Structure, function and folding of
                        Knotted Proteins
                        S. Jackson. Cambridge Univ.
4:50 PM        28       Protein folding Dynamics on and off the Ribosome
                        S. Cavagnero. Univ. of Wisconsin-Madison.

                                                                                      S. Cavagnero

                                    MONDAY, AUGUST 2
                        CONCURRENT AfTERNOON SYMPOSIA 8
                             MEMBRANE PROTEINS AND RECEPTORS
                                      2:45 PM—5:20 PM
                                  Douglas Pavilion A, 1st Level

Session Chair: Michael Feig, Michigan State Univ.
           Abstract #
2:45 PM        29       Lactose Permease
                        H. R. Kaback. UCLA.

3:15 PM        30       Exploring Integral Membrane Protein Structure and folding
                        C. Brooks. Univ. of Michigan.
3:45 PM        146      Young Investigator Talk
                        Transmembrane Protein Segments: Post-translational
                        Rearrangements and Integration into the Membrane of a-Helices
                        of Low Hydrophobicity L. Hedin. Stockholm Univ.
4:00 PM                 Break
4:20 PM        31       The Molecular function and Dysfunction of Membrane Attack
                        Complex/Perforin-Like Proteins
                        J. Whisstock. Monash Univ., Australia.
4:50 PM        32       Structure and Catalytic Mechanism of the E. coli Maltose
                        A. Davidson. Purdue Univ.
                                                                                        A. Davidson

              MONDAY, AUGUST 2

                     5:30 PM – 6:45 PM
           Exhibit Hall/Douglas Pavilion, 1st Level

                       WORKSHOP 4
              HOW TO GIVE A GREAT TALK
                   7:30 PM – 9:00 PM
                 Manchester D, 2nd Level
Ever sat through a boring talk and vowed never to give one?
This workshop offers clear guidelines on giving a great talk.
             Chair: Blake Hill, Johns Hopkins Univ.

                   NETWORKING DINNERS
                              7: 15 PM
Sign up on-site in the Registration Area, Douglas Foyer, 1st Level
                  first come, first served, self-pay
            Dinner locations to be announced on-site.

      7:30 AM – 5:00 PM
    Douglas Foyer, 1st Level

       7:30 AM – 6:30 PM
     Douglas Foyer, 1st Level

      7:30 AM – 8:30 AM
   Douglas Pavilion A, 1st Level

          8:00 AM – 6:45 PM
Exhibit Hall/Douglas Pavilion, 1st Level

                                    TUESDAY, AUGUST 3
                                        PLENARY SESSION 1
                                    fRED RICHARDS MEMORIAL
                                         9:00 AM—11:45 AM
                                      Douglas Pavilion A, 1st Level

Session Chair: TBD
           Abstract #
9:00 AM                 fred Richards and the Power of Quantitative Methods in Biology
                        F. Cohen. UCSF.
9:30 AM       33        The Structural Basis for P-TEfb (Cdk9/Cyclin T Recognition
                        L. Johnson. Univ. of Oxford.
10:00 AM                Break (refreshments in Exhibit Hall)
10:45 AM      34        fred Richards, Structural Biology, and the Ribosome
                        P. Moore. Yale Univ.
11:15 AM      35        Calmodulin: Bound Structures
                        F. Quiocho. Baylor Col. of Med.                                  P. Moore

                                           10:00 AM – 10:45 AM
                                  Exhibit Hall/Douglas Pavilion, 1st Level

                                            EXHIBITS OPEN
                                           10:00 AM – 6:45 PM
                                  Exhibit Hall/Douglas Pavilion, 1st Level

              TUESDAY, AUGUST 3
                       WORKSHOP 5
                       CAREER PANEL
This panel will help young scientists make decisions on pathways
        that may lead to industrial or academic careers.
                       12:00 PM – 1:00 PM
                     Manchester G, 2nd Level
             Chair: Blake Hill. Johns Hopkins Univ.

                 EXHIBITOR WORKSHOPS
                    12:00 PM – 1:00 PM
                  See page 63 for full listing.

                     POSTER SESSION 2
                       1:15 PM – 2:45 PM
             Exhibit Hall/Douglas Pavilion, 1st Level

                       2:00 PM – 2:45 PM
             Exhibit Hall/Douglas Pavilion, 1st Level

                                     TUESDAY, AUGUST 3
                         CONCURRENT AfTERNOON SYMPOSIA 9
                                 MICROfLUIDICS AND MICROARRAYS
                                          2:45 PM—5:20 PM
                                      Douglas Pavilion A, 1st Level

Session Chair: James Berger, Univ. of California, Berkeley.
            Abstract #
2:45 PM         36       Transcription factor-DNA Interactions: Cis Regulatory Codes in
                         M. Bulyk. Brigham and Women’s Hosp., Harvard Med. Sch.
3:15 PM         37       A Massively-Parallel Microchemostat Platform for Single Cell
                         S. Maerkl. Ecole Polytech. Fed. de Lausanne.                       M. Bulyk

3:45 PM         216      Young Investigator Talk
                         A Protein-Based Artificial Retina: Optimization of Chemically
                         Oriented Bacteriorhodopsin films for Electrical Stimulation of
                         Retinal Cells
                         M. Ranaghan. Univ. of Connecticut.
4:00 PM                  Break
                                                                                           S. Maerkl
4:20 PM         38       Electro-optical Trapping of Single Molecules at the 1 Nanometer
                         A. Cohen. Harvard Univ.
4:50 PM         39       Probing the Mechanical Response of Actin Networks with Atomic
                         force Microscopy
                         D. Fletcher. Univ. of California, Berkeley.

                                                                                            A. Cohen

                                                                                           D. Fletcher

                                     TUESDAY, AUGUST 3
                         CONCURRENT AfTERNOON SYMPOSIA 10
                                            2:45 PM—5:20 PM
                                         Manchester ABC, 2nd Level

Session Chair: Janet Smith, Univ. of Michigan.
            Abstract #
2:45 PM        40        Structure and Mechanism of Bacterial Transition Metal Sensing
                         Transcriptional Regulators
                         D. Giedroc. Indiana Univ.
3:15 PM        41        Single-Molecule Dynamics of Metal Regulators and Transporters
                         P. Chen. Cornell Univ.
3:45 PM        109       Young Investigator Talk
                         Mechanisms of Cu, zn Superoxide Dismutase Misfolding Induced
                         by Hydrogen Peroxide: Implications for Amyotrophic Lateral
                         V. Mulligan. Univ. of Toronto.
                                                                                           C. Drennan
4:00 PM                  Break
4:20 PM        42        Crystallographic Snapshots of Metalloenzyme Complexes Involved
                         in Biological Carbon Dioxide Sequestration
                         C. Drennan. MIT.
4:50 PM        43        How Aliphatic Halogenases Prevent Competing Hydroxylation
                         J. Bollinger, Jr. Penn State.
                                                                                          J. Bollinger, Jr.

                    TUESDAY, AUGUST 3

                            5:30 PM – 6:45 PM
                  Exhibit Hall/Douglas Pavilion, 1st Level

                         NETWORKING DINNERS
                                    7: 15 PM
       Sign up on-site in the Registration Area, Douglas Foyer 1st Level
                        first come, first served, self-pay
                  Dinner locations to be announced on-site.

                             WORKSHOP 6
                         7:30 PM—9:00 PM
                       Manchester D, 2nd Level
This workshop features “tricks of the trade” to best present your application
             for a postdoctoral position in a top laboratory.
                   Chair: Blake Hill, Johns Hopkins Univ.

      7:30 AM – 5:00 PM
    Douglas Foyer, 1st Level

       7:30 AM – 4:00 PM
     Douglas Foyer, 1st Level

          8:00 AM – 3:15 PM
Exhibit Hall/Douglas Pavilion, 1st Level

         10:00 AM – 3:30 PM
Exhibit Hall/Douglas Pavilion, 1st Level

         10:00 AM – 10:45 AM
Exhibit Hall/Douglas Pavilion, 1st Level

                                WEDNESDAY, AUGUST 4
                          CONCURRENT MORNING SYMPOSIA 11
                                PROTEIN DESIGN AND EVOLUTION
                                       9:00 AM—12:15 PM
                                    Douglas Pavilion A, 1st Level

Session Chair: Stephen Mayo, Caltech.
           Abstract #
9:00 AM        44       The Evolution of New Protein folds by Straightforward
                        Mutational Mechanisms
                        M.H. J. Cordes. Univ. of Arizona.
9:30 AM        45       In Vivo and in Vitro Evolution of an Anti-human IL-13 Antibody
                        J. Fransson. Centocor R&D, Johnson & Johnson, San Diego.
                                                                                         M.H. J. Cordes
10:00 AM                Break (refreshments in Exhibit Hall)
10:45 AM                Creating Novel Proteins and Assemblies by Selection and Design
                        L. Regan. Yale Univ.
11:15 AM       165      Young Investigator Talk
                        Transplantation of a Complex Binding Site Using Computational
                        Design and in Vitro Evolution
                        B. Correia. Univ. of Washington and New Univ. of Lisbon.
11:30 AM       537      Young Investigator Talk
                        Design of a Switchable Kemp Elimination Catalyst
                        I. Korendovych. Univ. of Pennsylvania.
11:45 AM       46       Structure-Guided SCHEMA Recombination Generates Highly
                        Stable and Functionally Diverse Enzyme Families
                        F. Arnold. Caltech.
                                                                                           F. Arnold

                                 WEDNESDAY, AUGUST 4
                          CONCURRENT MORNING SYMPOSIA 12
                                          AMYLOIDS AND DISEASE
                                             9:00 AM—12:15 PM
                                           Manchester ABC, 2nd Level

Session Chair: Carol Post, Purdue Univ.
           Abstract #
9:00 AM        47       Structural Studies of the Amyloid State
                        D. Eisenberg. UCLA-DOE Inst. for Genomics and Proteomics.
9:30 AM        48       folding and Aggregation of ALS-Associated Mutant Cu,zn,
                        Superoxide Dismutases
                        E. Meiering. Univ. of Waterloo, Canada.
10:00 AM                Break (refreshments in Exhibit Hall)
10:45 AM       49       Determination of the Structure of an Amyloid Protofibril with
                        Magic Angle Spinning NMR
                        R. Griffin. MIT.
11:15 AM       50       Membrane Binding and Aggregation of Alpha-Synuclein in
                        Parkinson’s Disease
                        J-C. Rochet. Purdue Univ.
11:45 AM       51       Adapting Proteostasis to Ameliorate Loss- and Gain-of-function
                        J. Kelly. The Scripps Res. Inst.                                 J. Kelly

                   WEDNESDAY, AUGUST 4
                        GRADUATE PROGRAM fAIR
                            12:15 PM – 1:45 PM
                         Manchester Foyer, 2nd Level
    Come learn more about the programs offered at these institutions and more!
                               Univ. of Minnesota
                         Univ. of Texas Medical Branch
                                Univ. of Montana
                           Cold Spring Harbor Lab
                               Univ. of Pittsburgh
                         Univ. of California, San fran
                                Univ. of Arizona
                         Univ. of Wisconsin Integrated
                                 U. C. San Diego

                          EXHIBITOR WORKSHOP
                             12:30 PM – 1:30 PM
                           See page 64 for full listing.

                               WORKSHOP 7
                      12:30 PM—1:30 PM
                     Manchester G, 2nd Level
Tools for clear scientific writing--demonstrated by deconstructing/reconstructing
                      an abstract from solicited participants.
                      Chair: Blake Hill, Johns Hopkins Univ.

                             POSTER SESSION 3
                               1:45 PM – 3:15 PM
                     Exhibit Hall/Douglas Pavilion, 1st Level

                               2:00 PM – 2:45 PM
                     Exhibit Hall/Douglas Pavilion, 1st Level

                                  WEDNESDAY, AUGUST 4
                                            1:30 PM—3:10 PM
                                         Manchester ABC, 2nd Level

Session Cochairs: Lisa Gentile, Univ. of Richmond; Ellis Bell, Univ. of Richmond.
            Abstract #
1:30 PM         508      Young Investigator Talk
                         Pharmacophore Modeling Strategies for the Development of Novel
                         Nonsteroidal Inhibitors of Human Aromatase (CYP19)
                         Y. Muftuoglu. Johns Hopkins Univ.
1:50 PM         549      Young Investigator Talk
                         Influence of the Hydrophobic Core on the Internal Friction of
                         folding: Experimental Studies of the four-Helix Bundle Protein
                         Rop with Wild-Type and Designed Cores
                         C. Cheng. Yale Univ.
2:10 PM         550      Young Investigator Talk
                         Ligand-Dependence Stability and folding in Cusf, a Bacterial
                         Copper(I) Chaperone
                         S. Ly. California State Univ., Channel Islands.
2:30 PM         569      Young Investigator Talk
                         The Varied Roles of Highly Conserved Residues near or within
                         Beta-Turns in Glyoxysomal Malate Dehydrogenase
                         S. Hedrick. Univ. of Richmond.
2:50 PM         440      Young Investigator Talk
                         Development of a Beta-Hairpin Model to Study the Role of PolyQ
                         Repeats in Cross-Beta-Sheet Aggregation.
                         A. Cuesta. Haverford Col.

                                WEDNESDAY, AUGUST 4
                                 AWARDS PLENARY SESSION 2
                                           3:20 PM—6:20 PM
                                        Manchester ABC, 2nd Level

Session Chair: TBD
           Abstract #
3:20 PM                 Opening Remarks
3:25 PM                 The Irving Sigal Young Investigator Award 2009
                        (sponsored by Merck Research Laboratories)
                        Presenter: James M. Schaeffer, Merck Research Laboratories.
3:30 PM       52        Expanding the Synthetic Capabilities of the Cell
                                                                                           V. Cornish
                        V. Cornish. Columbia Univ.
4:00 PM                 The Irving Sigal Young Investigator Award
                        (sponsored by Merck Research Laboratories)
                        Presenter: James M. Schaeffer, Merck Research Laboratories.
4:05 PM       53        Structural and Dynamic Basis for the Assembly of Protein
                        Machineries by NMR
                        C. Kalodimos. Rutgers Univ., Piscataway.
                                                                                          C. Kalodimos
4:35 PM                 Break (refreshments outside room)
5:05 PM                 The Stein and Moore Award (sponsored by The Protein Society)
                        Presenter: Daniel Raleigh (SUNY at Stony-Brook), President, The
                        Protein Society
5:10 PM       54        Excited States and (NMR) Relaxation
                        P.E. Wright. The Scripps Res. Inst.
5:40 PM                 The Dorothy Crowfoot Hodgkin Award (sponsored by Genentech)        P.E. Wright
                        Presenter: Wayne Fairbrother, Genentech.
5:45 PM       55        from Reductionist to Post-reductionist: A Protein folding
                        L. Gierasch. Univ of Massachusetts Amherst.
6:15 PM                 Closing Remarks

                                                                                          L. Gierasch

                   MANCHESTER ABC, 2ND LEVEL
              BANQUET fROM 8:00 PM – 9:30 PM

Speaker: Baldomero Olivera, Univ. of Utah, Distinguished Professor of Biology

                  Recognition of the following Awardees:
           Recognition of the Undergraduate Student Presentations
Presentation of Awards for Best Student Posters (sponsored by Eli Lilly & Co.)

                                  THURSDAY, AUGUST 5
                                        PLENARY SESSION 2
                                  MACROMOLECULAR MACHINES
                                       9:00 AM—12:00 PM
                                     Manchester ABC, 2nd Level

Session Chair: Todd Yeates, UCLA.
           Abstract #
9:00 AM        56       Grabbing the Cat by the Tail: Discrete Steps by a DNA Packaging
                        Motor and the Inter-subunit Coordination in a Ring-ATPase
                        C. Bustamante. Univ. of California, Berkeley.
9:30 AM        57       Dynamics and Stability in Virus Maturation: Harnessing a
                        Molecular Machine
                        J. Johnson. The Scripps Res. Inst.                                C. Bustamante

10:00 AM       295      Young Investigator Talk
                        Out-of-Equilibrium Conformational Cycling of GroEL Under
                        Saturating ATP Concentrations
                        G. Frank. Weizmann Inst. of Sci., Rehovot.
10:15 AM                Break (refreshments outside room)
10:45 AM       493      Young Investigator Talk
                        How prl Mutations or Binding of the Signal Peptide Unlock the      J. Johnson
                        Protein Translocon
                        A.N. Bondar. Univ. of California, Irvine.
11:00 AM       58       Structural Biology of Type IV Secretion Systems
                        G. Waksman. Inst. of Struct. and Molec. Biol., London, U.K.
11:30 AM                Molecular Machines That Remodel RNA: Translocation,
                        Unwinding and RNA folding by Sf2 Proteins
                        A. Pyle. Yale Univ., HHMI.

                                 POSTER SESSION 1
                             Monday, August 2, 2010
                     Exhibit Hall/Douglas Pavilion, 1st Level

                                                  Poster Set Up Time
                                                  7:00 AM – 8:00 AM

                                                 Poster Viewing Time
                                                  8:00 AM – 6:45 PM

                                         Author Presentations
                             Odd Board Numbers            1:15 PM – 2:00 PM
                             Even Board Numbers           2:00 PM – 2:45 PM

                                                   Poster Removal
                                                  6:45 PM – 7:15 PM

IMPORTANT: Please remove posters promptly at 6:45 PM so authors for the next session can place
         their posters on the Board. We are not responsible for lost poster boards.

         Sessions                                                                                                 Board Numbers
         Amyloids and Protein Misfolding............................................................B1 – B12
         Applications of Mass Spectrometry to Biological Problems ...................B13 – B16
         Computational Biology ............................................................................B17 – B21
         Cytoskeleton ............................................................................................B22
         Enzyme Kinetics and Mechanism ............................................................B23 – B29
         Imaging/Biosensors..................................................................................B30 – B38
         Intracellular Trafficking and Secretion ....................................................B39 – B41
         Membrane Proteins ..................................................................................B42 – B50
         New and Developing Methods.................................................................B51 – B59
         Protein Design and Protein Engineering ..................................................B60 – B69
         Protein Folding: General Aspects ............................................................B70 – B83
         Protein Networks .....................................................................................B84
         Protein–Nucleic Acid Interactions ...........................................................B85 – B92
         Protein Structure/Function Studies ..........................................................B93 – B116
         Protein–Based Biomaterials .....................................................................B117 – B120
         Protein–Protein and Protein–Ligand Interactions ....................................B121 – B146
         Receptors, Signaling and Signal Transduction ........................................B147 – B162
         Single Molecule Studies ..........................................................................B164 – B170

                                        MOnDAy,	AUGUST	2

Amyloids and Protein                                                 Applications of Mass
Misfolding                                                           Spectrometry to Biological
B1       100      Dynamic Local Unfolding in Alpha-1                 Problems
Antitrypsin Provides a Mechanism for Serpin
Polymerization B. Krishnan and L. Gierasch. Univ. of                 B13      112      Possibilities and Pitfalls in Quantifying
Massachusetts Amherst.                                               the	 Extent	 of	 Cysteine	 Sulfenic	 Acid	 Modification	 of	
B2       101      Repeat-Length Dependent Nucleus Size               Specific	Proteins	within	Complex	Biofluids D. Rehder and
and Polyglutamine Aggregation Kinetics K. Kar, R. Kodali             C. Borges. Arizona State Univ.
and R. Wetzel. Univ. of Pittsburgh Sch. of Med.                      B14      113      Proteomic Analysis of 3T3-LI Adipocyte
B3       102	     Expression	 and	 Purification	 of	 Growth	         Mitochondria during Differentiation, Maturation, and
Factor Receptor from Inclusion Bodies V. Badwaik,                    Hypertrophic Enlargement W. Russell, S. Cologna, B.
J. Bartonjo, J. Evans, C. Wills, A. Jain, S. Sahi and R.             Newton, A. Jayaraman and D. Russell. Texas A&M Univ.
Dakshinamurthy. Western Kentucky Univ.                               B15      114      Mass Spectrometry Reveals a Subunit
B4       103	     Rearrangement	 Reaction	 at	Asn-Gly	 of	           Interaction	 Map	 of	 the	 Phosphorylase	 Kinase	 Complex
GroES	Enhances	a	Conformation	That	Leads	to	Amyloid	                 O. Nadeau, L. Lane, C. Robinson and G. Carlson. Univ. of
Fibril Formation in Early Step H. Iwasa, S. Meshitsuka, K.           Kansas Med. Ctr., Univ. of Cambridge and Univ. of Oxford.
Hongo, T. Mizobata and Y. Kawata. Tottori Univ., Japan.              B16      115	     Chemical	 Cross-Linking	 and	 Mass	
B5       104	     Biophysical	     Characterization	      of	        Spectrometry for Structural Analysis of the Metal-
Fibrillogenic Fragments of Human Prion Protein J.                    Resistance	Protein	CusB	and	Determination	of	the	CusB/
Yau and S. Sharpe. Hosp. for Sick Children and Univ. of              CusF	Interaction	Site T. Mealman, V.H. Wysocki and M.M.
Toronto.                                                             McEvoy. Univ. of Arizona.
B6       105      Low Density Lipoprotein Inversely
Modulates	Amylin	Aggregation	and	Toxicity	in	Cultured	               Computational Biology
Rat	Beta-Cells S. Trikha and A. Jeremic. George Washington
Univ.                                                                B17      116	      Rigorous	Approximation	of	Continuum	
B7       106	     Characterization	 and	 Identification	 of	         Electrostatics:	The	Impact	of	Fast	Algorithms	and	GPU	
Amyloid Disaggregases A. Murray, J. Solomon, Y-J. Wang,              Computing J.P. Bardhan, R. Yokota, M.G. Knepley and
T. Mu, W. Balch and J. Kelly. The Scripps Res. Inst.                 L.A. Barba. Rush Univ. Med. Ctr., Univ. of Bristol, Univ. of
B8       107      Probing         Unique         Structural          Chicago and Boston Univ.
Characteristics	 between	 Different	 Prion	 Disease	 Strains	        B18      117	      Mechanism	 of	 Sliding	 Clamp	 Opening	
via	 Chemical	 Crosslinking	 and	 Mass	 Spectrometry M.              by	 the	 Clamp	 Loader	 RFC:	 Insight	 from	 Atomistic	
Moen and M. McGuirl. Univ. of Montana.                               Simulations I. Ivanov, J.A. McCammon and J. Tainer.
B9       108	     Characterization	 of	 Prion	 Protein	              Georgia State Univ., UCSD and The Scripps Res. Inst.
Multimers	Formed	under	Mildly	Acidic	Conditions X. Qi                B19      118	      An	 Energy-Sorted	 Conformer	 Library	
and M. McGuirl. Univ. of Montana.                                    Improves	Side	Chain	Sampling	in	Protein	Modeling	and	
B10      109	     Mechanisms	 of	 Cu,	 Zn	 Superoxide	               Design A. Senes and S. Subramaniam. Univ. of Wisconsin-
Dismutase Misfolding Induced by Hydrogen Peroxide:                   Madison.
Implications for Amyotrophic Lateral Sclerosis V.                    B20      119       Binding Free Energy Analysis Based on
Mulligan, P. Sharda, K. Hadley and A. Chakrabartty. Univ. of         GPGPU	Cluster M. Sekijima, K. Misoo, K. Ohno and M.
Toronto.                                                             Orita. Tokyo Inst. of Technol., Info. and Math. Sci. Lab. Inc.,
B11      110      In Vitro and in Vivo Aggregation of                Tokyo and Astellas Pharma Inc., Tokyo.
Mutants of a b-Clam	Protein M. Ferrolino, A. Zhuravleva, Z.          B21      120	      Inter-chain	 Disulfide	 Scrambling	 in	
Ignatova, A. Clouser and L. Gierasch. Univ. of Massachusetts         Human	 IgG2	 Antibodies:	 An	 Atomistic	 Insight	 from	
Amherst and Univ. of Potsdam, Germany.                               Molecular Dynamics Simulations X. Wang, S. Singh and S.
B12      111      Shifting the Edge: Protein Function                Kumar. Pfizer Inc., Chesterfield, MO.
Determines Protein Solubility Requirements P. Ciryam,
G.G. Tartaglia, M. Vendruscolo and C.M. Dobson. Univ. of             Cytoskeleton
Cambridge and Northwestern Univ.
                                                                     B22     121     Role of Dimerization in Talin Function
                                                                     A. del Rio, P. Roca-Cusachs and M.P. Sheetz. Columbia

                                          MOnDAy,	AUGUST	2
                                                                       B35      134     New DyLight 680 “Brighter” Far-Red
Enzyme Kinetics and                                                    Dye for Labeling and Detection B. Dworecki, M. Nlend, S.
                                                                       Desahi, J. Narahari and S. Shiflett. Thermo Fisher Scientific,
Mechanism                                                              Rockford, IL.
B23       122	   Allosteric	 Communication	 between	                   B36      135     Nanobody-Based Biosensor Detecting
Subunits	of	the	Dimeric	Phosphofructokinase-2	of	E. coli               Metastasizing	 Breast	 Cancer	 Cells M. Malecki and
Analyzed by Hybrid Enzymes J. Babul and M. Baez. Univ.                 R. Malecki. Western Univ. of Hlth. Sci. and NVI, San
of Chile.                                                              Francisco.
B24       123	   Revisiting	 the	 Catalytic	 Mechanism	 of	            B37      136     A Three-Hybrid Split-Luciferase Sensor
Triosephosphate	 Isomerase	 Using	 Site-Specific	 Mutants              for	Profiling	Protein	Kinases B. Jester, A. Gaj, C. Shomin,
M. Samanta and P. Balaram. Indian Inst. of Sci., Bangalore.            K. Cox, P. Wang and I. Ghosh. Univ. of Arizona.
B25       124	   Probing	the	Active	Site	of	Cystathionine	             B38      137	    An	 Autoinhibited	 Coiled-Coil	 Design	
Beta-Lyase P. Lodha and S. Aitken. Carleton Univ., Canada.             Strategy for Split-Protein Protease Sensors: Application
B26       125    Mutation Analysis of the Violaxanthin                 to	Caspases S. Shekhawat, J. Furman, J. Porter, A. Sriprasad
De-epoxidase	 Identifies	 Substrate	 Binding	 Site	 and	               and I. Ghosh. Univ. of Arizona.
Residues	 Involved	 in	 Catalysis G. Saga, A. Giorgetti, C.
Fufezan, G.M. Giacometti, R. Bassi and T. Morosinotto.
Univ. of padua, Univ. of Verona and Westfälische Wilhelms-
                                                                       Intracellular Trafficking and
Univ. Münster.                                                         Secretion
B27       126	   Examining	FXIII	Specificity	by	Assessing	
                                                                       B39      138      Polyubiquitin Recognition by Tandem
the	 Kinetic	 Contributions	 of	 Individual	 Residues	 in	
                                                                       Ubiquitin Binding Domains of Rabex-5 S.Y. Lee, D.H.
Glutamine-Containing	 Substrates P. Doiphode and M.
                                                                       Shin and S. Lee. Sungkyunkwan Univ., South Korea.
Maurer. Univ. of Louisville.
                                                                       B40      139      Protease and Hemolysin Production by
B28       127    Interaction of the Prolyl Isomerase Pin1
                                                                       Urogenital Isolates of E. faecalis D. Coombs, P. Porter and
with a Multi-phosphorylated Substrate A. Greenwood, S.
                                                                       L.W. Anderson. Westminster Col., UT.
De and L. Nicholson. Cornell Univ.
                                                                       B41      140      A pH-Regulated Folding Switch in
B29       128    Elucidation of the Pin1-APP Interaction
                                                                       Pseudomonas syringae Effector Protein AvrPto Modulates
Using NMR Lineshape Analysis S. De, E. Kovrigin and L.
                                                                       Its Translocation via the Type III Secretion System J.
Nicholson. Cornell Univ. and Med. Col. of Wisconsin.
                                                                       Seckute, J. Dawson, B. Kvitko, A. Collmer and L. Nicholson.
                                                                       Cornell Univ. and Colorado State Univ.
B30       129	    A	Bioorthogonal	Chemistry	Approach	to	               Membrane Proteins
Visualizing	Proteins	in	Live	Mammalian	Cells Q. Lin, W.
                                                                       B42      141      NMR Studies of the Structure, Dynamics,
Song, Z. Yu, Z. Wang and R. Lim. Univ. at Buffalo, SUNY.
                                                                       and	 Interactions	 of	 CXCR1	 and	 IL-8 F. Casagrande, L.
B31       130	    Construction	      of	    an	    Improved	
                                                                       Cho, L. Albrecht, M. Chu, S.H. Park and S. Opella. UCSD.
Optogenetic	 Chloride	 Sensor	 Using	 Cell-Free	 Protein	
                                                                       B43      142	     Preparative	 Scale	 Cell-Free	 Production	
Engineering Techniques J. Grimley, W. Wang, L. Li, L.
                                                                       of	G-Protein	Coupled	Receptors	in	High	Quality F. Junge,
Beese, G. Augustine and H. Hellinga. Duke Univ.
                                                                       C. Roos, V. Doetsch and F. Bernhard. Goethe Univ. Frankfurt/
B32       131     Directed Evolution of Protein-Based
                                                                       Main, Germany.
Neurotransmitter Sensors for Functional MRI E. Brustad,
                                                                       B44      143      An In-Membrane Hydropathy Scale
F. Bi, V. Lelyveld, A. Jasanoff and F. Arnold. Caltech and
                                                                       Developed	 with	 CFTR	 Constructs C. Mulvihill and C.
                                                                       Deber. Hosp. for Sick Children and Univ. of Toronto.
B33       132     A Nanoarray-Based Immunobiosensor
                                                                       B45      144      Over-Expression        and     Biophysical
for	 the	 Detection	 of	 an	 Antibody	 Secreted	 by	 a	 B-Cell	
                                                                       Characterisation	of	Membrane	Proteins	Solubilised	in	a	
Hybridoma N. Gulzar, D. Hohertz, C. Hui, R. Nirwan,
                                                                       Styrene	Maleic	Acid	Copolymer Y. Lin, R. Pacheco-Gomez,
S. Romanuik, S. Grist, R. Gordon, B. Gray, A. Brolo, K.
                                                                       Y-j. Yu, H-w. Huang, M. Jamshad, T. Knowles, C. Smith, M.
Kavanagh and J. Scott. Simon Fraser Univ. and Univ. of
                                                                       Overduin and T. Dafforn. Sch. of Biosci. and Sch. of Cancer
Victoria, Canada.
                                                                       Sci., Univ. of Birmingham and Univ. of Warwick, U.K.
B34       133     Synthesis of Targeted Fluorescent
                                                                       B46      145	     Binding	 Pocket	 Analysis	 of	 Seven	
Probes	for	Super-Resolution	Cellular	Imaging	of	Protein	
                                                                       Helical Transmembrane Proteins: Is Sequence Identity
Function N. Fu, Y. Xiong, C. Boschek, U. Mayer and T.
                                                                       Alone	Suitable	for	Modeling	GPCR	as	Drug	Targets? V.
Squier. Pacific Northwest Natl. Lab., Richland, WA.
                                                                       Pabuwal and Z. Li. Univ. of Sci. in Philadelphia and Univ. of

                                        MOnDAy,	AUGUST	2
B47      146       Transmembrane Protein Segments:                   B59     158	     Crystallizing	 Proteins	 by	 Synthetic	
Post-translational Rearrangements and Integration into               Symmetrization G. Forse, N. Ram, M. Sawaya, T. Yeates
the Membrane of a-Helices of Low Hydrophobicity L.                   and D. Cascio. UCLA.
Hedin, K. Öjemalm, A. Kauko, A. Hennerdal, K. Illergård,
I. Nilsson, G. von Heijne and A. Elofsson. Stockholm Univ.
and Åbo Akademi Univ., Finland.
                                                                     Protein Design and Protein
B48      147	      Expression	 and	 Purification	 of	 Active	        Engineering
State	Urotensin-II	Receptor	with	Affinity	Ligands A. Du,
L. Pattenden, M. Aguilar and W. Thomas. Monash Univ.,                B60       159      Antibody Fragment Engineering with
RMIT Univ. and Univ. of Queensland, Australia.                       Non-canonical Amino Acids J. Van Deventer, T.H. Yoo and
B49      148	      Identification	 and	 Purification	 of	            D. Tirrell. Caltech.
Heliobacterium modesticaldum NADH:menaquinone                        B61       160      Toward the Design of Self-Assembling
Oxidoreductase H. Yue and R. Blankenship. Washington                 Peptide	 Modules	 Supporting	 Cell-Attachment	 and	
Univ. in St. Louis.                                                  Growth B. Angelo, C. Pullar, P.M. Cullis and M.G. Ryadnov.
B50      149       Recapitulating the Native Local                   Univ. of Leicester and Natl. Physical Lab., London, U.K.
Environment of Transmembrane Segments with Detergent                 B62       161	     Flexible	 Backbone	 Design	 to	 Optimize	
Micelles D. Tulumello and C. Deber. Hosp. for Sick Children          2F5 mAb Epitope Transfer to Heterologous Proteins M.
and Univ. of Toronto.                                                Azoitei, J-P. Julien, Y-E. Ban, A. Schroeter, O. Kalyuzhniy,
                                                                     Y. Adachi, S. Bryson, D. Baker, E. Pai and W. Schief. Univ.
                                                                     of Washington, Univ. of Toronto and Ontario Cancer Inst./
New and Developing Methods                                           Princess Margaret Hosp., Toronto.
B51      150	    Modeling	 of	 Protein	 Complexes	 Using	            B63       162	     Stabilization	 of	 Cyanovirin-n,	 a	 Potent	
Filamentary Density Distributions A. Hausrath, A. Goriely            HIV-Inhibiting	 Lectin:	 Computer-Aided	 Design	 and	
and T-S. Tsao. Univ. of Arizona and Univ. of Oxford.                 Experimental	 Characterization V. Patsalo, D.P. Raleigh
B52      151     Development of a Multiplex ELISA Using              and D. Green. Stony Brook Univ.
Quantum Dots and Microarray Technology S. Schenk, K.                 B64       163      Towards a Molecular Understanding
Yau and T. Glancy. Dow AgroSciences, Indianapolis.                   of Protein Solubility R. Kramer, S. Trevino, C.N. Pace and
B53      152     Thermodynamic Analysis of Self                      J.M. Scholtz. Texas A&M Hlth. Sci. Ctr.
Assembly	 in	 Coiled-Coil	 Biomaterials B. Kokona, B.                B65       164      Oligonucleotide Recombination for
Tsang, H. Bretscher and R. Fairman. Haverford Col.                   Combinatorial	Optimization	of	yeast	Metabolic	Flux	for	
B54      153	    Coupling	 Somatic	 Hypermutation	 with	             Terpenoid Production N. Ostrov, P.K. Ajikumar, Z. Fowler,
Mammalian	 Cell	 Surface	 Display:	 A	 novel	 Approach	              M. Koffas, G. Stephanopoulos and V. Cornish. Columbia
to the Discovery and Optimization of Therapeutic                     Univ., MIT and Univ. at Buffalo, SUNY.
Antibodies R. Toobian, T. Neben, H. Jones, J. Dalton, I.             B66       165	     Transplantation	 of	 a	 Complex	 Binding	
Krapf, G. Tomlinson, J. Macomber, A. Chen, L. Altobell III,          Site	Using	Computational	Design	and	in	Vitro	Evolution
T. Holland, R. O’Hanlon, B. Wu, R. Horlick, D. King and P.           B. Correia, M. Azoitei, Y-E. Ban, O. Kalyuzhniy, A. Schroeter,
Bowers. AnaptysBio Inc., San Diego.                                  D. Baker and W. Schief. Univ. of Washington and New Univ.
B55      154	    Tuning	 the	 Stability	 of	 Green	                  of Lisbon.
Fluorescence Protein Promotes Mutational Robustness                  B67       166	     Structural	Analysis	of	Computationally	
for Non-canonical Amino Acids Incorporation N.                       Designed	 Kemp	 Elimination	 Catalysts T.M. Lee, H.K.
Soundrarajan, G. Raghunathan and S-G. Lee. Pusan Natl.               Privett and S.L. Mayo. Caltech and Protabit LLC, Pasadena.
Univ., South Korea.                                                  B68       167	     Reengineering	        Small	       GTPase	
B56      155	    Gold	 Cluster	 Labeling	 as	 a	 new	 Tool	          Interactions	 Using	 Computational	 Design	 to	 Rewire	
to Study Protein Structures Using Small Angle X-Ray                  Protein Signaling Pathways S. Liu, G. Kappa, N. Ollikainen
Scattering H.C. Lee, B. Craig, C. Bailey-Kellogg, C. Le and          and T. Kortemme. UCSF.
A. Friedman. Purdue Univ. and Dartmouth Col.                         B69       168	     Towards	 Design	 of	 Species-Specific	
B57      156	    An	 Ultra	 High-Efficient	 System	 for	 in	         Snake	 Toxins O. Sharabi, G. Dakwar, Y. Aizner and J.
Vivo and in Vitro Synthesis of Tyrosine-Sulfated Proteins            Shifman. The Hebrew Univ. of Jerusalem.
L-Y. Lu, B-H. Chen, J. Wu, C-C. Wang, T-H. Lin, C-C. Hsu,
C-C. Lin and Y-S. Yang. Natl. Chiao Tung Univ., Taiwan.
B58      157     Self Assembly of TMAO at Hydrophobic
Interfaces and Its Effect on Protein Adsorption: Insights
from Experiments and Simulations G. Anand, S. Jamadagni,
S. Garde and G. Belfort. Rensselaer Polytech Inst.

                                        MOnDAy,	AUGUST	2
                                                                    B81      180      Beyond the Test Tube: Monitoring
Protein Folding: General                                            Protein Folding in Vivo L. Foit, A. Mueller, T. Tadesse, G.
                                                                    Morgan, M. Kern, A. von Hacht, L. Steimer, J. Titchmarsh,
Aspects                                                             S. Warriner, S. Radford and J. Bardwell. Univ. of Michigan,
B70      169      FoldEco: Simulating Protein Folding               Univ. of Leeds and HHMI, Chevy Chase, MD.
in E. coli K. Pobre, E. Powers and L. Gierasch. Univ. of            B82      181      Kinship of n→p* Interactions and
Massachusetts Amherst and The Scripps Res. Inst.                    Hydrogen Bonds A. Choudhary and R.T. Raines. Univ. of
B71      170      OxidativeAssembly of the Octadecameric            Wisconsin-Madison.
Complex	 of	 an	 Adipocyte	 Hormone	 Requires	 Oxygen	              B83      182      Two Salivary Lipocalins from Blood-
and	Stabilization	of	Assembly	Intermediates	by	Disulfide	           Sucking	Insects	Have	Different	Folds	Despite	Significant	
Bonds	 —	 A	 Model	 for	 Assembly	 of	 Protein	 Complexes	          Sequence Similarity C. Roessler and M.H. J. Cordes. Univ.
with	 Short	 Collagen	 Domains T-S. Tsao, D. Briggs, M.             of Arizona.
Nunez and A. Hausrath. Univ. of Arizona.
B72      171	     Folding	the	Circular	Permutants	of	IL-
1b: A Balancing Act between Topological Symmetry and
                                                                    Protein Networks
Functional Heterogeneity D. Capraro, S. Gosavi, M. Roy, J.          B84     183	     A	 Computational	 Protocol	 for	
Onuchic and P. Jennings. UCSD and Natl. Ctr. for Biol. Sci.,        Quantifying	 Interaction	 Specificity	 between	 Protein	
TIFR, Bangalore.                                                    Families L. Au and D. Green. Stony Brook Univ.
B73      172	     nMR	and	CD	Analysis	of	an	Intermediate	
State in the Thermal Unfolding Process of Mouse
Lipocalin-Type Prostaglandin D Synthase Y. Miyamoto,
                                                                    Protein–Nucleic Acid
Y. Noda, S. Nishimura, S-i. Segawa and T. Inui. Grad. Sch.          Interactions
of Life and Envrn. Sci., Osaka Prefect. Univ., Japan Soc.
for Promotion of Sci., Tokyo and Sch. of Sci. and Technol.,         B85      184	       Metal	 Selectivity	 in	 the	 Ferric	 Uptake	
Kwansei Gakuin Univ., Japan.                                        Regulator M. Dominguez and S. Mills. Univ. of San Diego.
B74      173	     Physical-Chemical	Determinants	of	Coil	           B86      185        Interactions of the E. coli Primosomal
Conformations	 in	 Globular	 Proteins L. Perskie and G.             PriB Protein with the Single-Stranded DNA: Solution
Rose. Johns Hopkins Univ.                                           versus	 Crystallographic	 Studies M. Szymanski, M.
B75      174      Investigation of the Thermal Stability of         Jezewska and W. Bujalowski. Univ. of Texas Med. Branch.
a Y-Family DNA Polymerase S. Sherrer, L. Pack, K. Fiala,            B87      186        Kinetics of Protein Translocation
J. Fowler and Z. Suo. The Ohio State Univ.                          between Two DNA Duplexes at Equilibrium: Validation
B76      175	     Structural	 Characterization	 of	 the	            of NMR Studies D. Sahu and J. Iwahara. Univ. of Texas
Sub-millisecond	 Refolding	 Intermediate	 of	 Chey S.               Med. Branch.
Kathuria, O. Bilsel, P. Nobrega and C.R. Matthews. Univ. of         B88      187        The N-Terminal Domain of Murine
Massachusetts Med. Sch.                                             Hepatitis	 Virus	 nucleocapsid	 Protein	 Binds	 Specifically	
B77      176	     Slipknotting	 upon	 native-Like	 Loop	            to the Transcriptional Regulatory Sequence and Plays
Formation in a Trefoil Knotted Protein J. Noel, J.                  a	 Critical	 Role	 in	 Viral	 Replication S. Keane, P. Liu, J.
Sulkowska and J. Onuchic. UCSD.                                     Leibowitz and D. Giedroc. Indiana Univ. and Texas A&M
B78      177      Probing the Structure of the H2A-H2B              Hlth. Sci. Ctr. Col. of Med.
Dimeric Kinetic Folding Intermediate by Site-Directed               B89      188        Role of a Novel Domain in Splicing
Mutagenesis P. Guyett and L. Gloss. Washington State                Factor 1 during the Early Stages of Pre-mRNA Splicing
Univ.                                                               A. Gupta and C.L. Kielkopf. Univ. of Rochester.
B79      178	     Dissecting	 the	 CL	 Domain	 of	                  B90      189        Nucleotide Analogs and Molecular
Streptococcal	Collagen-Like	Protein	2 Z. Yu, B. Brodsky             Modeling Studies Reveal Key Interactions Involved in
and M. Inouye. UMDNJ-Robert Wood Johnson Med. Sch.                  Substrate Recognition by the Yeast RNA Triphosphatase
and UMDNJ-Grad. Sch. of Biomed. Sci.                                M. Issur, S. Despins, I. Bougie and M. Bisaillon. Univ. of
B80      179	     The	 Possible	 Folding	 Zone	 within	 the	        Sherbrooke, Canada.
E.coli Ribosomal Exit Tunnel K-F. Lin, C-S. Sun, Y-C.               B91      190        Streptococcus pneumoniae NmlR Is a
Huang and J-T. Huang. Inst. of Chem. and Inst. of Biomed.           Single	 Cysteine	 Containing	 nitric	 Oxide	 Sensor	 That	
Sci., Acad. Sinica, Taipei.                                         Forms a Stable S-NO Adduct G. Campanello and D.
                                                                    Giedroc. Indiana Univ.
                                                                    B92      191	       Structural	 Flexibility	 of	 the	 Cytidine	
                                                                    Repressor DNA-Binding Domain C. Moody, V.
                                                                    Tretyachenko-Ladokhina, D.F. Senear and M.J. Cocco. Univ.
                                                                    of California, Irvine.

                                        MOnDAy,	AUGUST	2
                                                                     B104 203          Structure of the Mycobacterium
Protein Structure/Function                                           tuberculosis Virulence Factor Rv0899(OmpATb) Y. Yao,
                                                                     P. Teriete, A. Kolodzik, J. Yu, H. Song, M. Niederweis and
Studies                                                              F. Marassi. Sanford Burnham Med. Res. Inst., La Jolla and
B93      192	      Defining	 the	 Interaction	 between	              Univ. of Alabama at Birmingham.
S100A13 and Annexin II Peptide: Insight into Non-                    B105 204	         Calmodulin	 Activation	 of	 Estrogen	
classical Secretion A. Daily, A. Martfeld and T.K. S. Kumar.         Receptor Alpha R. Bieber Urbauer, C. Jolly, S. Johnson,
Univ. of Arkansas.                                                   J. Galdo, M. Cross, M. Elliott, M. Nooromid, L. Cho, N.
B94      193       Understanding the Structure and                   Cheung, B. Jones, E. Henderson and J. Urbauer. Univ. of
Stability	of	FGF-1	in	a	Cellular	Environment	Using	nMR	              Georgia and Bruker AXS Inc., Madison, WI.
Spectroscopy D. Vo and T.K. S. Kumar. Univ. of Arkansas.             B106 205          Molecular Determinants of Human
B95      194       The Role of Heparin in Fibroblast                 Beta-Defensin 3 Binding to Melanocortin Receptors 1 and
Signaling R. Kerr, N. Webb and T.K. S. Kumar. Univ. of               4 M. Nix, C. Kaelin, T. Ta, G. Barsh and G. Millhauser. Univ.
Arkansas.                                                            of California, Santa Cruz and Stanford Univ.
B96      195       Development of an S100A13 Monomer                 B107 206          Mechanism        of     Pollutant-Induced
in	 Order	 to	 Further	 Define	 the	 non-classical	 Release	         Conformational	Alterations	 in	 Human	 Serum	Albumin:	
Pathway	of	FGF-1 A. Martfeld, E. Erstine, T.K. S. Kumar              A	 Key	 to	 Catalytic	 Heterotropic	 Allostericity	 Causing	
and A. Daily. Univ. of Arkansas.                                     Non-microbial Drug Resistance E. Ahmad, G. Rabbani and
B97      196       Bioinformatics and Kinetic Studies in             R.H. Khan. Aligarh Muslim Univ., India.
Human Pyridoxal Kinase Reveal a New Motif Associated                 B108 207	         The	 Bifunctional	 Roles	 of	 nickel	 Ions	
to	Zinc	Coordination	and	Regulation	of	Enzyme	Activity               in Helicobacter pylori [NiFe]-Hydrogenase Maturation
V. Guixé and C. Ramírez. Univ. of Chile.                             Pathway W. Xia, H. Li, K-H. Sze and H. Sun. The Univ. of
B98      197       Structure of D-AKAP2-PKA RI Isoform               Hong Kong.
Complex:	Insights	into	AKAP	Specificity	and	Selectivity              B109 208          Structural          and         Functional
G. Sarma, F. Kinderman, C. Kim, S. von Daake and S. Taylor.          Characterization	of	a	novel	Homodimeric	Three-Finger	
UCSD, Amgen Inc., Baylor Col. of Med. and HHMI, La                   Neurotoxin from the Venom of Ophiophagus hannah
Jolla.                                                               (King	Cobra) A. Roy, X. Zhou, D. Dieter, M.Z. Chong, C.S.
B99      198	      Contact	 Surface	 Mapping	 of	 LysRS	 in	         Foo, N. Rajagopalan, S. Nirthanan, D. Bertrand, J. Sivaraman
Its Multi-functional States by Solution-Phase Hydrogen/              and R.M. Kini. Natl. Univ. of Singapore, Univ. of Geneva,
Deuterium	 Exchange	 FT-ICR	 Mass	 Spectrometry H-M.                 Sch. of Med. Sci., Griffith Univ., Australia and Virginia
Zhang, M. Guo, X-L. Yang, P. Schimmel, Q. Zhang, M.R.                Commonwealth Univ.
Emmett and A.G. Marshall. Natl. High Magnetic Field Lab.,            B110 209	         Cloning,	 Protein	 Purification,	 and	
Florida State Univ. and The Scripps Res. Inst.                       Functional	 Characterization	 of	 CutC,	 a	 Copper	
B100 199           Biochemical and Structural Studies of             Homeostasis Protein in Caenorhabditis elegans J. Yang,
HMW1C-Like	Glycosyltransferases H-J. Yeo, K. Choi, S.                C. Srinivasan and N. Nikolaidis. California State Univ.,
Paek and F. Kawai. Univ. of Houston.                                 Fullerton.
B101 200           Structure-Function Relationships for              B111      210     Structural and Functional Analysis of
Substrate Selectivity in 2-Hydroxy Acid Oxidizing                    Bacterial DNA Polymerase X Involved in DNA Repair
Enzymes S. Leitgeb, T. Stoisser, D. Neuhold and B. Nidetzky.         Processes S. Nakane, H. Ishikawa, N. Nakagawa, R. Masui
GmbH, Graz and Graz Univ. of Technol., Austria.                      and S. Kuramitsu. Osaka Univ. and RIKEN SPring-8 Ctr.,
B102 201	          High-Resolution	 Crystal	 Structures	 of	         Hyogo, Japan.
Intact	and	Cleaved	Forms	of	Bovine	Pancreatic	Trypsin	               B112 211	         Characterization	 of	 the	 n-Terminus	 of	
Inhibitor Variants Bound to Trypsin J. Penfield, E.                  Human	Copper	Transporter	(HCtr1) X. Du, X. Wang and
Zacharova, S. Classen, M.P. Horvath and D.P. Goldenberg.             H. Sun. The Univ. of Hong Kong.
Univ. of Utah and Lawrence Berkeley Natl. Lab.                       B113 212	         Crystal	     Structures	    of	    Human	
B103 202	          Systematic	Approach	to	Better	Crystals            Thioredoxin Revealing Unraveled Helix and Exposed
Q. Zhao, Q. Wang, S. Chen, T. Dong, X. Liu, X. Hou, Y.               S-Nitrosation Site J. The, A. Weichsel, M. Kem and W.
Zhai, S. Liu, Y. Liu, F. Xiao, Y. Han, H. Hou, M. Liu, C. Qi,        Montfort. Univ. of Arizona.
Y. Wang, Q. Huang, C. Ke, Y. Sun, Q. Wang, M.Y. Xia and              B114 213	         Disease-Causing	 Missense	 Mutations	
Z.M. Wang. Crown Bioscience Inc., Beijing.                           in Actin Binding Domain 1 of Dystrophin Induce
                                                                     Thermodynamic Instability and Protein Aggregation
                                                                     D. Henderson, A. Lee and J. Ervasti. Univ. of Minnesota,

                                          MOnDAy,	AUGUST	2
B115 214          Probing Potential Protein Dynamics                   B124 223           Understanding the Potentiation of
to Elucidate the Mechanism for Enzymatic Activity in                   nMDA	 iGluRs	 by	 Pregnenolone	 Sulfate K. Cameron, S.
Human	 Immunodeficiency	 Virus	 Type-1	 Protease S.                    Rhoads and L. Gentile. Univ. of Richmond.
Mittal and C. Schiffer. Univ. of Massachusetts Med. Sch.               B125 224           Mutational and Structural Analysis of
B116 215	         Crystallization	 of	 the	 Pbx1-Pdx1-DnA	             the	P2’	Specificity	of	Mesotrypsin	toward	Kunitz	Protease	
Complex	on	a	Somatostatin-Specific	Promoter	Sequence                   Inhibitors M. Salameh, A. Soares and E. Radisky. Mayo
D. Wang and R. Rose. North Carolina State Univ.                        Clin. Cancer Ctr., Jacksonville and Brookhaven Natl. Lab.
                                                                       B126 225	          Genome-Wide	 Structural	 Modeling	 of	
                                                                       Protein-Protein Interactions P. Kundrotas, Z. Zhu and I.
Protein-Based Biomaterials                                             Vakser. Univ. of Kansas.
B117 216	          A	 Protein-Based	 Artificial	 Retina:	              B127 226           Modeling of Protein-Protein Interactions
Optimization	of	Chemically	Oriented	Bacteriorhodopsin	                 by Structural Similarity at the Interfaces P. Kundrotas, R.
Films	 for	 Electrical	 Stimulation	 of	 Retinal	 Cells M.             Sinha and I. Vakser. Univ. of Kansas.
Ranaghan, N. Wagner, M. Sandberg, D. Sandberg, R. Grewal,              B128 227           Thermodynamic Basis of Nucleotide
R. Rangarajan, A. Nellisery, R. Jensen and R. Birge. Univ. of          Discrimination	in	the	PBCV-1	mRnA	Capping	Enzyme:	
Connecticut and VA Boston Healthcare Syst.                             A Thermodynamic Integration Study R. Swift and R.E.
B118 217           Preparation of Liposomal Nanovesicles               Amaro. Univ. of California, Irvine.
with	Influenza	Hemagglutinin	Activity M-Y. Wang, G.R-L.                B129 228	          Identification	 of	 the	 Antibody	 Phage	
Chang, S-y. Lai, P-T. Chu and H-W. Wen. Natl. Chung                    Display-Derived	Fab25	as	a	Potent	and	Specific	Inhibitor	
Hsing Univ. and Central Taiwan Univ. of Sci. and Technol.,             of Serine Protease Hepsin R. Ganesan, Y. Zhang, P. Moran,
Taiwan.                                                                S. Sidhu and D. Kirchhofer. Genentech.
B119 218           Drug Delivery System for Poorly Water-              B130 229           Structural      and       Thermodynamic
Soluble Drugs by Lipocalin-Type Prostaglandin D                        Determinants of Noncovalently Oligomeric Protein
Synthase A. Fukuhara, H. Nakajima, K. Inoue, Y. Miyamoto,              Ligands G. Poon and A. Forbes. Washington State Univ.
S. Shimamoto, S. Nishimura, T. Ohkubo, T. Takeuchi and T.              B131 230           Improvement of Hit Ratio Using
Inui. Grad. Sch. of Life and Envrn. Sci., Osaka Prefect. Univ.,        Combination	 of	 in	 Silico	 and	 SPR	 Screening	 for	 Anti-
Japan Soc. for Promotion of Sci., Tokyo, Japan Synchrotron             prion	Compounds H. Nakamura, Y. Kamatari, J. Hosokawa-
Radiation Res. Inst., Hyogo and Grad. Sch. of Pharmaceut.              Muto and K. Kuwata. Ctr. for Emerging Infect. Dis., Gifu,
Sci., Osaka Univ.                                                      Japan.
B120 219	          Biological	 Protein	 Crystals:	 A	 novel	           B132 231           Hit Validation by Direct Measurement of
Platform	 for	 Cellular	 Imaging	 and	 Protein	 Delivery M.            Protein	Conformational	Change M. Gostock, J. Popplewell,
Nair, M. Lee, J. Wallace, R. Burry, M. Ostrowski and M.                G. Ronan and M. Swann. Fafield Group, Pittsburgh and
Chan. The Ohio State Univ.                                             Crewe, U.K.
                                                                       B133 232           The Production of Protein-Protein
                                                                       Complexes	 in	 High-Throughput	 Manner	 for	 Functional	
Protein-Protein and Protein-                                           and	Structural	Characterization G. Babnigg, R. Jedrzejczak,
Ligand Interactions                                                    B. Nocek, A. Stein, W. Eschenfeldt, N. Marshall, A. Weger,
                                                                       R. Wu, L. Stols, K. Buck and A. Joachimiak. Argonne Natl.
B121 220	         Comparing	      Two	      Methods	     for	          Lab.
Determining	 the	 Binding	 Affinity	 of	 an	 Antibody	 to	 a	          B134 233           Application      of      a    Biophysical
Cell	Surface	Expressed	Target S. Scesney, L. Kalghatgi, E.             Characterization	 Platform	 in	 Developing	 Chemical	
Barlow, M. Leddy, S. Zhong, X. Lu and D. Karaoglu. Abbott              Probes G. Senisterra, A. Allali Hassani, A. Siarheyeva, G.
Bioresearch Ctr., Worcester.                                           Wasney, I. Chao, T. Hajian, P. Brown, C. Arrowsmith and M.
B122 221	         Specificity	 and	 Cooperativity	 at	                 Vedadi. Univ. of Toronto.
b-Lactamase Position 104 in TEM-1/BLIP and SHV-1/                      B135 234	          Energetics-Based	 Target	 Identification	
BLIP Interactions M. Hanes, K. Reynolds, C. McNamara,                  by Prefractionation and Pulse Proteolysis C. Park and Y.
P. Ghosh, R. Bonomo, J. Kirsch and T. Handel. Skaggs Sch.              Chang. Purdue Univ.
of Pharm., UCSD, Univ. of Texas Southwestern Med. Ctr.,                B136 235	          The	 Binding	 Affinity	 of	 Cyanovirin-n	
UCSD, Louis Stokes VA Med. Ctr., Cleveland and Univ. of                to	Viral	Glycoproteins	Is	Correlated	with	the	number	of	
California, Berkeley.                                                  Functional Binding Sites I. Maier, A. Berry, J. Keeffe, S.K.
B123 222          Understanding the Inhibitory Regulation              Gillespie and S.L. Mayo. Caltech.
of	AMPA	and	nMDA	iGluRs	by	Sulfated	neurosteroids
K. Cameron, D. Fanelli, B. Pollard, E. Bartle and L. Gentile.
Univ. of Richmond.

                                         MOnDAy,	AUGUST	2
B137 236            Interplay       between         Reaction          B149 248           Structural Basis of Shh Regulation by
Stoichiometry	and	Effective	Concentration:	A	Structure-               Neutralizing Antibody 5E1 and Hedgehog-Interacting
Based	Synergetic	Study	on	Grb2-Sos1	Complex A. Sethi,                 Protein: Role of the Shh Pseudo Active Site in Signaling
B. Goldstein and S. Gnanakaran. Los Alamos Natl. Lab.                 H. Maun, I. Bosanac, S.J. Scales, X. Wen, A. Lingel, J.F.
B138 237            Small Molecule Inhibitor Studies with             Bazan, F.J. de Sauvage, S.G. Hymowitz and R.A. Lazarus.
the	 Human	 Cytoplasmic	 Protein	 Tyrosine	 Phosphatase	              Genentech Inc.
and Structural Elucidation of Its Protein Target, EphA2               B150 249           Dissecting      Phototaxis      in      the
G. Costakes, K. Homan, D. Balasubramaniam, O. Wiest, P.               Cyanobacterium	 Synechocystis	 sp.	 Strain	 PCC6803
Helquist and C. Stauffacher. Purdue Univ. and Notre Dame              S. Wisén, D. Levy and D. Bhaya. Carnegie Instn. for Sci.,
Univ.                                                                 Stanford and Univ. of Maryland College Park.
B139 238            Quantitative      Measurement           of        B151 250           The MyD88:IRAK4:IRAK2 Death
Dephosphorylation of EphA2 by Both Isoforms                           Domain	Complex	Reveals	a	Hierarchical	Helical	Oligomer	
of	 the	 HCPTP	 Using	 SRM	 Mass	 Spectrometry D.                     in TLR/IL1-R Signaling S-C. Lin, Y-C. Lo and H. Wu. Weill
Balasubramaniam, L. Paul, K. Homan, M. Hall and C.                    Cornell Med. Col.
Stauffacher. Purdue Univ.                                             B152 251	          Genetically	 Engineered	 scFv	 against	
B140 239	           Coupled	 Folding	 and	 Binding	 of	 the	          Stage-Specific	 Embryonic	 Antigens	 Promote	 Isolation	
MLL Translocation Partners AF4 and AF9 Is Required                    of	Pluripotent	Bone	Marrow	Stem	Cells M. Malecki and
for	 Transcriptional	 Elongation	 in	 Acute	 Leukemia B.              B. Malecki. Western Univ. of Hlth. Sci. and PBMEC, San
Leach, T. Cierpicki, N. Zeleznik-Le and J. Bushweller. Univ.          Francisco.
of Virginia, Univ. of Michigan and Loyola Univ. Med. Ctr.             B153 252	          Role	 of	 Gtr1-Gtr2	 Complex	 Formation	
B141 240	           Understanding	             Carbohydrate	          on Their Biologic Function T. Sekiguchi, Y. Kamada, Y.
Recognition	 by	 the	 Antiviral	 Lectin	 Cyanovirin-n Y.              Ohsumi, Y. Wang and H. Kobayashi. Grad. Sch. of Med. Sci.,
Fujimoto and D. Green. Stony Brook Univ.                              Kyushu Univ., Natl. Inst. for Basic Biol., Okazaki, Tokyo
B142 241	           Expression	and	Purification	of	Mutated	           Inst. of Technol., NICHD, NIH and Okayama Univ.
Hpn Protein, and Studies of Interaction between UreE                  B154 253	          Enhanced	Expression	of	Cation	Channel	
and Hpn/Hpnl S. Qi. The Univ. of Hong Kong.                           of	Sperm	(CatSper)	in	GC-1	spg	Cells	by	Ginseng	Radix	
B143 242            Micropatterning of Plasma Membrane                Treatment E.H. Park, M.S. Chang, S.K. Park and W.M. Yang.
Proteins	 to	Analyze	 Raft	 Localization	 in	 Living	 Cells S.        Col. of Oriental Med., Kyung Hee Univ., South Korea.
Sunzenauer, J. Weghuber, M. Brameshuber, L. Rajendran and             B155 254           Interaction of 18-Methoxycoronaridine
G. Schuetz. JKU Linz, Austria and Univ. of Zurich.                    with Nicotinic Acetylcholine Receptors in Different
B144 243	           Re-scoring	 Protein-Protein	 Docking	             Conformational	 States K. Dinwiddie, A. Rosenberg, D.
Decoys	by	Characterizing	Energy	Landscape K. Park and                 Feuerbach, K. Targowska-Duda, K. Jozwiak, S. Glick, R.
D. Kim. KAIST, Daejeon, South Korea.                                  Moaddel, I. Wainer and H. Arias. Col. of Pharm., Midwestern
B145 244            Investigation of Thrombin Interactions            Univ., AZ, NIA, NIH, Baltimore, Novartis Insts. for Biomed.
with ABE-I Ligands Using HDX and MALDI-TOF Mass                       Res., Basel, Med. Univ. of Lublin, Poland and Albany Med.
Spectrometry M. Malovichko and M. Maurer. Univ. of                    Col.
Louisville.                                                           B156 255           Understanding the Molecular Basis
B146 245            Acyl-Homoserine Lactone Induces                   of Protein-Peptide Interaction by the Two Tandem
a	 Conformational	 Change	 of	 the	 Quorum	 Sensing	                  Protein Tyrosine Phosphatase Domains of Drosophila
Transcriptional Regulator SdiA M. Gallardo, N. Guiliani,              RPTPs DLAR and DPTP99A L. Madan, S. Veeranna, K.
P. Prevelige, Jr. and O. Monasterio. Univ. of Chile and Univ.         Shameer, R. Sowdhamini and B. Gopal. Indian Inst. of Sci.,
of Alabama at Birmingham.                                             Bangalore.
                                                                      B157 256           Taiwan Banded Krait Protease Inhibitor-
                                                                      Like	 Protein-1-Induced	 Apoptosis	 of	 Human	 Leukemia	
Receptors, Signaling and                                              U937	 Cells	 through	ADAM17/Lyn/Akt	 Pathways H. Liu
Signal Transduction                                                   and L-S. Chang. Natl. Sun Yat-Sen Univ., Taiwan.
                                                                      B158 257	          Systematic	 Analysis	 of	 Human	 PDZ	
B147 246          Phosphorylation-Dependent                           Domain-Ligand	 Interaction	 network	 and	 Their	
Deactivation of Melanopsin J. Blasic and P. Robinson.                 Localization J. Kim, J-s. Yang, S. Park, Y.S. Choi, J. Jeon, J.
Univ. of Maryland Baltimore County.                                   Hwang and S. Kim. POSTECH, South Korea.
B148 247          Phosphorylation Status of Kinases and               B159 258	          Computational	Models	Provide	Insights	
Its Implications in Drug Discovery K. Ray, J. Sikora, J. Yu           into	Heterotrimeric	G-Protein	Function N. Carrascal and
and B-S. Pan. Merck Res. Labs., Boston.                               D. Green. Stony Brook Univ.

                                        MOnDAy,	AUGUST	2
B160 259	         Conformation	 and	 Activity	 of	 the	              B166 265         Selective RNA Annealing and Unwinding
Intracellular Domain of the Arabidopsis	 CRInKLy4	                   Mechanisms of Hfq Revealed by Single-Molecule
Receptor-Like	Kinase M. Meyer and A.G. Rao. Iowa State               Measurements W. Hwang, V. Arluison and S. Hohng. Seoul
Univ.                                                                Natl. Univ. and Univ. Paris 7.
B161 260	         Over-Expression	 of	 Calcium	 Buffer	              B167 266         Unfolding and Refolding Dynamics of
Protein	 Delays	 Calcium	 Signaling	 and	 Disrupts	 Store-           Filamin	A	Protein	Under	Constant	Forces H. Chen, X. Zhu,
Operated	Calcium	Entry E. Pham, E. Mills and K. Truong.              F. Nakamura, M.P. Sheetz and J. Yan. RCE in Mechanobiol.,
Univ. of Toronto.                                                    Singapore, Natl. Univ. of Singapore, Harvard Med. Sch.,
B162 261	         Periplasmic	 Sensing	 of	 Cu(I)/Ag(I)	 by	         Brigham and Women’s Hosp. and Columbia Univ.
CusS S. Aravind, C. Rensing and M.M. McEvoy. Univ. of                B168 267         Untying Knots in Proteins J. Sulkowska,
Arizona.                                                             P. Sulkowski, P. Szymczak, J. Onuchic and M. Cieplak.
                                                                     UCSD, Caltech, Warsaw Univ. and Polish Acad. of Sci.,
Single Molecule Studies                                              B169 268	        Engineering	 Cysteine	 Residues	 into	
B164 263	           Dynamic	 Bending	 and	 Cleavage	 of	 a	          Glyoxysomal	 Malate	 Dehydrogenase	 to	 Study	 Subunit	
DnA	Gate	by	Human	Topoisomerase	IIa S. Hohng, S. Lee                 Interactions Using Fluorescence and Atomic Force
and N. Osheroff. Seoul Natl. Univ. and Vanderbilt Univ. Sch.         Microscopy Approaches M.J. Drake and E. Bell. Univ. of
of Med.                                                              Richmond.
B165 264	           Investigations	 on	 Conformational	              B170 269         Imaging of Mobile Stable Lipid-
Heterogeneity of Ubiquitin by spFRET L-L. Yang, M.W-P.               Mediated	 Protein	 Associations	 in	 the	 Live	 Cell	 Plasma	
Kao, H-L. Chen, T-S. Lim, W. Fann and R.P-Y. Chen. Inst.             Membrane M. Brameshuber, V. Ruprecht, J. Weghuber, I.
of Phys., Inst. of Biol. Chem. and Inst. of Atomic and Molec.        Gombos, I. Horvath, L. Vigh, H. Stockinger and G. Schuetz.
Sci., Acad. Sinica, Taipei and Tunghai Univ., Taiwan.                Univ. of Linz, Austria, Biol. Res. Ctr., Hungarian Acad. of
                                                                     Sci., Szeged and Med. Univ. of Vienna.

                                 POSTER SESSION 2
                             Tuesday, August 3, 2010
                     Exhibit Hall/Douglas Pavilion, 1st Level

                                                 Poster Set Up Time
                                                 7:00 AM – 8:00 AM

                                                Poster Viewing Time
                                                 8:00 AM – 6:45 PM

                                        Author Presentations
                            Odd Board Numbers            1:15 PM – 2:00 PM
                            Even Board Numbers           2:00 PM – 2:45 PM

                                                   Poster Removal
                                                  6:45 PM – 7:15 PM

IMPORTANT: Please remove posters promptly at 6:45 PM so authors for the next session can place
         their posters on the Board. We are not responsible for lost poster boards.

         Sessions                                                                                                Board Numbers
         Bioinformatics, Genomics, Proteomics ...................................................B1 – B6
         Chromatography and Protein Purification ...............................................B7 – B14
         Computational Biology ............................................................................B15 – B17
         Enzyme Kinetics and Mechanism ............................................................B18 – B21
         Membrane Proteins ..................................................................................B22 – B25
         Molecular Machines: Function and Assembly .........................................B26 – B31
         Peptide Biochemistry and Protein-Peptide Interactions ..........................B32 – B39
         Post-translation Modification of Proteins ................................................B40 – B47
         Protein Design and Protein Engineering ..................................................B48 – B57
         Protein Disorder .......................................................................................B58 – B64
         Protein Dynamics: Theory and Experiment .............................................B65 – B75
         Protein Folding: Mechanisms ..................................................................B76 – B91
         Protein–Nucleic Acid Interactions ...........................................................B92 – B99
         Protein Quality Control ............................................................................B100 – B101
         Protein Structure Prediction .....................................................................B102 – B109
         Protein Structure/Function Studies ..........................................................B110 – B133
         Protein Therapeutics and Targets .............................................................B134 – B154
         Protein-Protein and Protein-Ligand Interactions .....................................B155 – B167
         Receptors, Signaling and Signal Transduction ........................................B168 – B170

                                         TUESDAy,	AUGUST	3
                                                                      B11       280     Estimating Protein Molecular Weight
Bioinformatics, Genomics,                                             Using the Extended Range Prestained Precision Plus
                                                                      Protein Dual Xtra Standards J. Siino, S. Lin, J. Kohn, D.
Proteomics                                                            Yee and M. Urban. Bio-Rad Labs., Hercules, CA.
B1       270	     Application	 of	 Graph-Theoretical	                 B12       281	    HPLC	 Fluorescent	 Micelle	 Assay	 for	
Approach and Nonlinear Dynamics to Protein Structure                  Accurate	Determination	of	Polysorbate	80	Concentration	
Representation, Analysis and Prediction M. Pesenson, A.               in Therapeutic Protein Formulations C. Chen, A. Vaidya,
Kister and I. Pesenson. Caltech, Rutgers Univ., Piscataway            T. Estey, L. Salmeron and J. Siemiatkoski. PPDi, La Jolla and
and Temple Univ.                                                      Biogen Idec.
B2       271      Pan Phylum Analysis of Protein-Protein              B13       282	    novel	Purification	Protocol	for	Heparin	
Interactions Reveals Potential Drug Targets for Helminths             Binding Proteins S. Batra, N. Sahi, K. Mikulcik, H.
C. Taylor, S. Abubucker, Z. Wang, J. Martin, K. Fischer, P.           Shockley, C. Turner, E. Conte and R. Dakshinamurthy.
Fischer, D.J. Jiang, G. Weil and M. Mitreva. Washington               Western Kentucky Univ.
Univ. Sch. of Med.                                                    B14       283     Application      fo    Multi-dimensional
B3       272	     Proteomic	Analysis	of	Copper	Response	              Reversed-Phase	HPLC	for	the	Isolation	of	a	Calcitonin-
in E. coli A. George Thompson, G. Longoni and M.M.                    Like	Diuretic	Hormone	in	Tenebrio molitor D. Jensen and
McEvoy. Univ. of Arizona.                                             D.A. Schooley. Univ. of Nevada, Reno.
B4       273      A Novel Method for Protein-Protein
Interaction Site Prediction with Phylogenetic Substitution
Models D. La and D. Kihara. Purdue Univ.
                                                                      Computational Biology
B5       274	     Cysteine	Oxidation	Prediction	Program:	             B15      284     Ligand-Induced         Fit     Activation
A New Software Program That Predicts Reversible                       Mechanism	 of	 GH5	 Endoglucanase:	 A	 Molecular	
Protein	Cysteine	Thiol	Oxidation	Reactions R. Sanchez, L.             Dynamics Study S. Badieyan, D. Bevan and M. Zhang.
Grant and J. Momand. California State Univ., Los Angeles.             Virginia Tech.
B6       275      Local Sequence Hydropathy in Folded                 B16      285     A Protein Trajectory Reconstruction
Proteins I. Borukhovich and M.H. J. Cordes. Univ. of                  Strategy	 from	 FRET-Derived	 Distance	 Constraints A.
Arizona.                                                              Savol and C. Chennubhotla. Univ. of Pittsburgh.
                                                                      B17      286     Investigation of the Layered Structure of
                                                                      HIV-1 gp120 Using Temperature-Accelerated Molecular
Chromatography and Protein                                            Dynamics A. Emileh and C. Abrams. Drexel Univ.
B7       276	     Cloning,	 Expression,	 Purification	 and	           Enzyme Kinetics and
Functional	 Characterization	 of	 Recombinant	 Aleuria
aurantia Lectin L. Birkenmeyer, T. Rae, Q. Ruan, S. Diep,
C. Zhao, R. Ziemann, R. Bonn and A.S. Muerhoff. Abbott                B18      287	     Biochemical	 Characterization	 of	 the	
Labs.                                                                 Artificial	 S262T	 Mutant	 of	 the	 Metallo-Beta-Lactamase	
B8       277	     Bacterial	Co-expression	as	a	Convenient	            IMP-1 K. Pegg, A. George, K. Lee and P. Oelschlaeger. Cal
Method	 for	 the	 Production	 and	 Purification	 of	 Core	            Poly Pomona.
Histones R. Dutnall, M. Anderson, J. Huh, T. Ngo, A. Lee,             B19      288	     Computational	Approach	to	Investigate	
G. Hernandez, J. Pang and J. Perkins. Univ. of San Diego,             the	 Catalytic	 Chemical	 Reaction	 Mechanism	 of	
UCSD and San Diego State Univ.                                        Glyoxysomal	 Malate	 Dehydrogenase H. Guterres and E.
B9       278	     Cloning,	         Expression	           and	        Bell. Univ. of Richmond.
Characterization	of	Recombinant	Human	Hemopexin	in	                   B20      289	     The	 Effect	 of	 Molecular	 Crowding	 on	
HEK293	Cells B. Dille, L. Birkenmeyer, R. Coffey, Q. Ruan,            Allosteric	Kinetics	and	Allosteric	Regulation	of	Glutamate	
S. Saldana, S. Tetin, M. Pinkus, T. Rae and A.S. Muerhoff.            Dehydrogenase S. Asante and E. Bell. Univ. of Richmond.
Abbott Labs.                                                          B21      290	     Identification	 of	 Residues	 in	 the	
B10      279      An Amine Activated Agarose Support                  Propeptide of Pseudomonas aeruginosa Elastase with
for	 Quick	 and	 Efficient	 Immobilization	 of	 Proteins	 and	        Roles	in	Chaperone	Function E. Boice, E. Kessler and D.
Antibodies R. Ganapathy, M. Schofield, A. Deshpande and               Ohman. Virginia Commonwealth Univ., Tel Aviv Univ. Sheba
S. Desai. Thermo Fisher Scientific, Rockford, IL.                     Med. Ctr. and VA Med. Ctr., Richmond, VA.

                                       TUESDAy,	AUGUST	3
                                                                   B33      302	     Identification	of	Molecular	Determinants	
Membrane Proteins                                                  of Secretin for Binding and Activation of Its Receptor M.
                                                                   Dong, A. Le, D. Pinon, A. Bordner and L. Miller. Mayo Clin.,
B22      291	    A	 Computational	 Study	 of	 Translocon-
Assisted Insertion of Helical Proteins into Membranes A.
                                                                   B34      303      De Novo Design of Antimicrobial
Rychkova and A. Warshel. Univ. of Southern California.
                                                                   Peptides with Decreased Binding to Human Serum
B23      292	    GPCR	 Activation:	 Structural	 Insights	
                                                                   Proteins Z. Yan, S. Bevers, Z. Jiang, M. Vasil and R. Hodges.
from	 the	 CB1	 Cannabinoid	 Receptor C. Scott, R. Abrol
                                                                   Univ. of Colorado Sch. of Med., Aurora.
and W. Goddard III. Caltech.
                                                                   B35      304	     The	Proenkephalin	Prohormone	Displays	
B24      293     NMR Structural Studies of Mercury
                                                                   Intact Protein-Dependent Differential Accessibilities of
Transport Membrane Proteins W.S. Son, G. Lu, H.
                                                                   Multiple	Protease	Cleavage	Site	Domains	to	the	Aqueous	
Nothnagel, A. Davis and S. Opella. UCSD.
                                                                   Environment W. Lu, T. Liu, S. Li, V. Woods. Jr. and V. Hook.
B25      294     Structure and Dynamics of Vpu from
                                                                   Sch. of Pharm. and Sch. of Med., UCSD.
HIV-1 by NMR H. Zhang, Y. Wang, S.H. Park, L. Chan and
                                                                   B36      305	     Semi-synthesis	 of	 Glutamate	 Receptor-
S. Opella. UCSD.
                                                                   Associated Proteins—Structure and Function Relationship
                                                                   Study	 of	 PDZ-Domains S.W. Pedersen, A. Bach, A.
Molecular Machines: Function                                       Kristensen and K. Strømgaard. Univ. of Copenhagen.
                                                                   B37      306      Targeting HIV-1 Envelope Proteins
and Assembly                                                       Utilizing a Multivalent Approach P. Wang, M. Zhou, B.
B26      295	     Out-of-Equilibrium	     Conformational	          Wellensiek, N. Ahmad and I. Ghosh. Univ. of Arizona.
Cycling	of	GroEL	Under	Saturating	ATP	Concentrations               B38      307	     One-Bead	 One-Compound	 Combinato-
G. Frank, G. Haran and A. Horovitz. Weizmann Inst. of Sci.,        rial Peptide Libraries for Development of Effective
Rehovot.                                                           Strategies	 for	 Diagnosis	 of	 Prostate	 Cancer L. Meng,
B27      296      Discrimination of the Structure of the           F-M.H. Elbadri, F.Y. S. Chuang, A.L. Gryshuk, C.H.
Amino Acid by the Translational Machinery A. Olivo, J.             Contag, S.M. Lane and K.S. Lam. Univ. of California Davis,
Avins, P. Effraim, M. Englander, R. Fleisher, J. Wang, R.          Sacamento and Stanford Univ.
Gonzalez and V. Cornish. Columbia Univ.                            B39      308      Discovering Bivalent Ligands for Protein
B28      297	     The	Cytosolic	Domain	of	Mitochondrial	           Kinases via Phage Display Peptide Libraries C. Shomin, S.
Fission Protein, Fis1, Reversibly Binds and Inserts into           Meyer, E. Restituyo, V. Lamba, K. Cox and I. Ghosh. Univ.
Lipid Membranes to Affect Membrane Morphology R.                   of Arizona.
Wells, L. Picton, M. Koppenol, A. Mujumdar and R.B. Hill.
Johns Hopkins Univ.                                                Post-translation Modification of
B29      298      Mechanisms and Atomic Structures of
the Shell Proteins from a Bacterial Organelle in Salmonella        Proteins
enterica C. Crowley, J. Kopstein, F. Sheng, M. Sawaya, D.
                                                                   B40      309	     Context-Dependent	            n-Terminal	
Cascio, T. Bobik and T. Yeates. UCLA and Iowa State Univ.
                                                                   Acetylation of Recombinant Proteins in Escherichia coli
B30      299      Discrimination of the Structure of the
                                                                   Y. Ryu, L. Bernal-Perez, F. Sahyouni and L. Prokai. Texas
Amino Acid by the Translational Machinery R. Fleisher,
                                                                   Christian Univ. and Univ. of North Texas Hlth. Sci. Ctr.
J. Avins, P. Effraim, M. Englander, A. Olivo, J. Wang, R.
                                                                   B41      310	     OST3/6-Like	Thiol	Oxidoreductases	and	
Gonzalez and V. Cornish. Columbia Univ.
                                                                   Redox	Control	of	n-Linked	Protein	Glycosylation	in	the	
B31      300      The Ribosome Discriminates the
                                                                   ER P. Jutur and D. Fomenko. Univ. of Nebraska-Lincoln.
Structure of the Amino Acid at Its Peptidyltransferase
                                                                   B42      311      Production of Functionally Active
Center J. Avins, M. Englander, R. Gonzalez and V. Cornish.
                                                                   Glycoproteins	 and	 Phosphoproteins	 Using	 a	 Human	
Columbia Univ.
                                                                   in Vitro Expression System E. Hommema, G. Kilmer,
                                                                   K. Vattem, S. Mikami, K. Maas, M. Schofield, B. Webb,
Peptide Biochemistry and                                           A. Deshpande and H. Imataka. Thermo Fisher Scientific,
                                                                   Rockford, IL, RIKEN SSBC, Yokohama and Univ. of Hyogo,
Protein-Peptide Interactions                                       Japan.
B32      301      Molecular       Recognition      between         B43      312	     Glycosylation	 Stabilizes	 the	 Molten	
Individual	 Domains	 of	 Calmodulin	 and	 Target	 Binding	         Globule	 of	 Alpha-1	 Antitrypsin	 without	 Stabilizing	 the	
Sequences Involves a Kinetically Distinct Intermediate             Native State A. Sarkar and P.L. Wintrode. Case Western
State C. Boschek, Y. Xiong, D.M. Smith, C. Baird and T.            Reserve Univ.
Squier. Pacific Northwest Natl. Lab., Richland, WA.

                                         TUESDAy,	AUGUST	3
B44      313      TMEM16A/Anoctamin-1                Has a            B57      326	     Cystine-Knot	Peptides	Engineered	with	
Homodimeric Architecture G. Schmalzing, G. Fallah, S.                 Specificities	for	aIIb b3 or aIIb b3 and av b3 Integrins
Detro-Dassen, U. Braam and F. Markwardt. RWTH Aachen                  Are Potent Inhibitors of Platelet Aggregation M. Kariolis,
Univ. and Martin Luther Univ. Halle, Germany.                         A. Silverman and J. Cochran. Stanford Univ.
B45      314      Yeast Ribosomal Proteins Rpl12ap
and Rpl12bp Are Structurally Identical M. Tam and T.T.
Nguyen. Inst. of Molec. Biol., Acad. Sinica, Taipei.
                                                                      Protein Disorder
B46      315      A Facile Preparation of Ubiquitinated               B58      327      Effects of Intrinsic Disorder in
Proteins Using Pyrrolysine Analogs X. Li, T. Fekner, J.               Regulating the Balance between Intra- and Inter-
Ottesen and M. Chan. The Ohio State Univ.                             molecular Interactions in the Signaling Adapter NHERF-1
B47      316      Diversion of Post-translational Protein             H. Cheng, R. Fazlieva, A. Lewandowska and H. Roder. Fox
Sulfation in Plant and Other Organisms C-C. Hsu, L-Y.                 Chase Cancer Ctr.
Lu, B-H. Chen, T-H. Lin, C-C. Wang, J.Y-S. Wu, C-C. Lin               B59      328      Aquifex aeolicus FlgM Protein Exhibits
and Y-S. Yang. Natl. Chiao Tung Univ., Taiwan.                        a Temperature Dependent Disordered Nature M. Gage,
                                                                      R. Molloy, W.K. Ma, A. Allen, K. Greenwood, L. Bryan, R.
Protein Design and Protein                                            Sacora and L. Williams. Northern Arizona Univ.
                                                                      B60      329	     Characterization	 of	 the	 FGF	 Receptor	
Engineering                                                           Mutations Involved in Disease R. Thurman and T.K. S.
                                                                      Kumar. Univ. of Arkansas.
B48       317	    Stabilizing	      Cellulase:	    Sterically	        B61      330	     Conformational	 and	 Spectroscopic	
Conservative	 Charged-to-Uncharged	 Amino	 Acid	                      Characterization	 of	 Intrinsically	 Disordered	 Regions	 in	
Mutants	 of	 CelB2,	 the	 Beta-1,4-Endoglucanase	 from	               Proteins A. Sethi, D. Vu and S. Gnanakaran. Los Alamos
Streptomyces lividans N.A. Sawyer, E.K. Makwana and P.                Natl. Lab.
Kahn. Rutgers Univ.                                                   B62      331      Fibrin Resistance to Lysis in Patients with
B49       318     Prediction of B. circulans Xylanase                 Pulmonary Hypertension Other Than Thromboembolic
Mutations	That	Contribute	to	Increased	Thermostability                M. Becatti, M. Miniati, C. Fiorillo, B. Olimpieri, S. Monti,
P. Galberg, M.P. Rostkowski, M.H. M. Olsson, L.P. McIntosh,           M. Bottai, C. Marini, E. Grifoni, B. Formichi, C. Bauleo, C.
L.L. Leggio and J.H. Jensen. Univ. of Copenhagen and Univ.            Arcangeli, D. Poli, P. Nassi, R. Abbate and D. Prisco. Univ.
of British Columbia.                                                  of Florence, CNR, Pisa, Univ. of South Carolina Arnold Sch.
B50       319     Evolution      of    I-SceI   Derivatives           of Publ. Hlth. and Azienda Hosp., Univ. of Careggi, Italy.
Exhibiting	narrowed	Specificity R. Joshi, K. Tenney, K.K.             B63      332      Dimensionality Reduction Reveals
Ho, J-H. Chen, B. Golden and F. Gimble. Purdue Univ.                  Differences between Disordered Protein Dynamics and
B51       320     Engineered p-Stacking	 Interactions	 in	            Early-Stage Protein Folding Dynamics J. Phillips, E. Lau,
Monomeric Red Fluorescent Proteins M. Moore, R. Chica                 S. Newsam and M. Colvin. Univ. of California, Merced and
and S.L. Mayo. Caltech and Univ. of Ottawa.                           Lawrence Livermore Natl. Lab.
B52       321     A Molecular Dynamics Approach for                   B64      333      The Nuclear Localization Signal
Pre-screening	Computational	Enzyme	Design	Results B.                  Peptide of NF-kB(p65) Folds upon Binding to IkBa C.
Sosa Padilla Araujo, T. Miller III and S.L. Mayo. Caltech.            Cervantes, S-C. Sue, G. Kroon, M. Kierkgaard, E. Komives
B53       322	    Computational	 Protein	 Design	 of	                 and H.J. Dyson. UCSD, The Scripps Res. Inst. and Univ. of
Structural Ensembles: Validation by Automated                         Copenhagen.
Screening	of	Combinatorial	Libraries A. Nisthal, B. Allen
and S.L. Mayo. Caltech.
B54       323     Three Surface Mutations Produce a                   Protein Dynamics: Theory and
Novel Oligomer in Arc Repressor K.L. Holso, E.D. Dodds,
V.H. Wysocki and M.H. J. Cordes. Univ. of Arizona.
B55       324     Using Molecular Dynamics Simulations                B65     334       Plant Antimicrobial Peptide Thionin
to Predict Domain Swapping Y. Mou and S.L. Mayo.                      Forms a Water-Permeable Pore S. Oard and F. Enright.
Caltech.                                                              Louisiana State Univ.
B56       325	    Engineering	 High	 Affinity	 Knottin	               B66     335	      Backbone	 Dynamics	 of	 the	 Cleaved	
Peptides	 Targeting	 Tumor	 Marker	 CAIX	 for	 Cancer	                Form of Bovine Pancreatic Trypsin Inhibitor A. Lund, J.J.
Imaging and Therapy S. Moore and J. Cochran. Stanford                 Skalicky and D.P. Goldenberg. Univ. of Utah.
Univ.                                                                 B67     336	      Protein	 Conformational	 Changes	 Are	
                                                                      Derived	 from	 the	 network	 of	 Evolutionarily	 Coupled	
                                                                      Residues J. Jeon, Y-E. Shin, J. Hwang, Y.S. Choi, J-s. Yang,
                                                                      H-J. Nam and S. Kim. POSTECH, South Korea.

                                       TUESDAy,	AUGUST	3
B68       337     Molecular Flexibility and Antibacterial           B81      350	     Cysteine	Sulfenic	Acid	as	an	Intermediate	
Resistance: Functional Dynamics of the BlaR1 Sensor                 in	Disulfide	Bond	Formation	and	in	Vitro	Protein	Folding
Domain B. Wilson and J. Peng. Univ. of Notre Dame.                  C. Borges and D. Rehder. Arizona State Univ.
B69       338	    Investigating	 Conserved	 Motions	 in	            B82      351	     All-Atom	 Simulations	 of	 CSK	 Revels	
Proteins:	 Defining	 the	 Signaling	 Conduit	 in	 Pin1 K.           Thermodynamics and Kinetic Role of ATP during
Wilson, A. Namanja and J. Peng. Univ. of Notre Dame and             Functional Transitions L. de Oliveira, P. Whitford, P.
City of Hope Med. Ctr.                                              Jennings and J. Onuchic. UCSD and Los Alamos Natl. Lab.
B70       339     Dynamic Allostery in Proteins with Two-           B83      352      Protein     Stability,    Folding,    and
Fold Symmetry H. Toncrova and T. McLeish. Univ. of Leeds            Aggregation of N-Terminal Actin Binding Domains of
and Univ. of Durham, U.K.                                           Dystrophin and Utrophin K. Mallela, S. Singh, J. Molas, J.
B71       340	    What	 Can	 a	 Single	 Mutation	 Do? A.            Cabello-Villegas and N. Kongari. Univ. of Colorado Denver,
Goel, B. Tripet, R. Tyler, J. Carey and V. Copie. Montana           Aurora.
State Univ., Med. Col. of Wisconsin and Princeton Univ.             B84      353	     Origins	of	Backtracking H. Lammert, J.
B72       341	    Exploring	 Conformational	 Flexibility	           Noel and J. Onuchic. UCSD.
of Partially Folded States of Apomyoglobin with Site-               B85      354      Mapping         a      Native      Protein
Directed Spin Labeling C. Lopez, S. Oga and W. Hubbell.             Conformational	 Landscape	 to	 Reveal	 Transient	 Partial	
Geffen Sch. of Med. at UCLA.                                        Unfolding Events in Apomyoglobin D. Meinhold, H.J.
B73       342     Uncovering Order in Disorder: A                   Dyson and P.E. Wright. The Scripps Res. Inst.
Molecular Simulation Study of the Self-Assembly of                  B86      355	     GeoFold:	A	Mechanistic	Framework	for	
Elastin S. Rauscher and R. Pomès. Hosp. for Sick Children           Simulating Protein Unfolding C. Bystroff, V. Ramakrishnan,
and Univ. of Toronto.                                               S. Salem, M. Zaki, S. Srinivasan, S. Matthews and W. Colon.
B74       343	    Analysis	 of	 the	 Side-Chain	 Rotameric	         Rensselaer Polytech Inst., Inst. of Bioinformat. and Applied
States	 throughout	 the	 Enzymatic	 Cycles	 of	 E. coli and         Biotechnol., Bangalore, North Dakota State Univ. and Texas
human Dihydrofolate Reductase L. Tuttle, H.J. Dyson and             A&M Univ.
P.E. Wright. The Scripps Res. Inst.                                 B87      356	     Many	Folding	Paths	to	Green	Fluorescent	
B75       344	    Relaxation	 Dispersion	 Profiles	 of	 the	        Protein Y-m. Huang, S. Nayak, P. Reeder, J. Dordick and C.
Calcium-Binding	Proteins	S100A5	and	S100A1 M. Liriano,              Bystroff. Rensselaer Polytech Inst. and Univ. of Colorado at
K. Varney, R. Ishima and D. Weber. Univ. of Maryland Sch.           Boulder.
of Med. and Univ. of Pittsburgh Sch. of Med.                        B88      357	     Skp	 and	 SurA,	 Chaperones	 in	 the	
                                                                    Periplasim	 of	 Gram-negative	 Bacteria	 That	 Assist	 in	
                                                                    Outer Membrane Biogenesis C. Sandoval and M. Sousa.
Protein Folding: Mechanisms                                         Univ. of Colorado at Boulder.
B76       345     Laser-Induced        Propagation      and         B89      358	     Denatured	 Polypeptide	 Chains	 Can	
Destruction of Amyloid b Fibrils H. Yagi, D. Ozawa, K.              Adopt	Knotted	Conformations A. Mallam, J. Rogers and S.
Sakurai, T. Kawakami, H. Kuyama, H. Naiki and Y. Goto.              Jackson. Univ. of Cambridge and Univ. of Texas at Austin.
Osaka Univ. and Fukui Univ., Japan.                                 B90      359	     Geometric	 Frustration	 in	 the	 Folding	
B77       346     Folding and Function of Proteins from             Landscape of MitoNEET E. Baxter, J. Onuchic and P.
the b-Trefoil Fold Family S. Gosavi and J. Onuchic. Tata            Jennings. UCSD.
Inst. of Fundamental Res., Bangalore and UCSD.                      B91      360      Folding Initiation Sites in Polarized and
B78       347     Single Molecule Investigation of the              Diffuse Transition State Ensembles P. Buck and C. Bystroff.
Denatured	State	Dynamics	of	Cytochrome	c in the Time                Rensselaer Polytech Inst.
Domain from 100 ms to 1 S S. Takahashi, H. Oikawa and K.
Kamagata. Tohoku Univ., Japan.
B79       348     Probing Dynamics and Structure on the
                                                                    Protein–Nucleic Acid
Folding Energy Landscape of HisF TIM Barrel from T.                 Interactions
maritima	 by	 Chemical	 Denaturation	 and	 native-State	
Hydrogen Exchange G. Gangadhara, J.M. Laine, F. Massi               B92     361      Deduced RNA Binding Mechanism
and C.R. Matthews. Univ. of Massachusetts Med. Sch.                 of ThiI Y. Tanaka, S. Yamagata, Y. Kitago, Y. Yamada, S.
B80       349     A Potassium Switch of ATP-Induced                 Chimnaronk, M. Yao and I. Tanaka. Hokkaido Univ., Japan
GroEL	Conformational	Changes J. Chen, K. Makabe and                 and Mahidol Univ., Thailand.
K. Kuwajima. Natl. Insts. of Natural Sci., Okazaki and Grad.        B93     362	     HMGn2	 Binds	 to	 the	Acidic	 Region	 of	
Univ. for Adv. Studies, Japan.                                      nucleosomes	and	Competes	with	Histone	H1	for	Binding	
                                                                    Sites B-R. Zhou, H. Feng, H. Kato, M. Bustin and Y. Bai.
                                                                    NCI, NIH.

                                         TUESDAy,	AUGUST	3
B94      363     The Universal DNA Structure in                       B106 375          Molecular Mechanics and Dynamics
Homologous Recombination T. Mikawa, T. Masuda, Y.                     Based	Modeling	of	the	Major	Gas	Vesicle	Protein,	GvpA
Ito, T. Terada and T. Shibata. RIKEN, Yokohama, Tokyo                 H. Ezzeldin, J. Klauda, S. DasSarma and S. Solares. Univ. of
Metropolitan Univ. and Univ. of Tokyo.                                Maryland College Park and Univ. of Maryland Biotechnol.
B95      364     Determining the Role of PaxB in Porifera             Inst., Baltimore.
A. Rued, B. Cieniewicz, L. Gentile and A. Hill. Univ. of              B107 376          Improving Protein Sequence Alignment
Richmond.                                                             by	Using	Correlated	Mutation	Information C-s. Jeong and
B96      365     Proximity of the N-Terminus of the                   D. Kim. KAIST, Daejeon, South Korea.
Archaeal	 General	 Transcription	 Factor	 TFB1	 to	 DnA	              B108 377          A Novel High-Resolution Protein
during Transcription Initiation M. Bartlett and M.                    Structure	 Refinement	 Algorithm M. Chitsaz and S.L.
Micorescu. Portland State Univ. and Oregon Hlth. & Sci.               Mayo. Caltech.
Univ.                                                                 B109 378	         BCL::Fold,	 a	 novel	 de	 novo	 Protein	
B97      366	    Changes	 in	 the	 Kinetic	 Behavior	 of	 the	        Structure Prediction Method M. Karakas, N. Woetzel,
Zalpha-Induced	 B-Z	 Conversion	 by	 Introducing	 Single-             N. Alexander, S. Lindert, R. Staritzbichler and J. Meiler.
Stranded Regions Y-G. Kim and H.J. Park. Sungkyunkwan                 Vanderbilt Univ.
Univ., South Korea.
B98      367     Functional Role of Arg106 Involved in
RnA-Binding	of	Human	Coronavirus	OC43	nucleocapsid	
                                                                      Protein Structure/Function
Protein M-H. Hou and I-J. Chen. Chung-Hsing Univ.,                    Studies
B99      368	    Effect	 of	 Trigger	 Loop	 Conformation	             B110 379	         The	 Conformational	 and	 Functional	
on RNA Polymerase II Dynamics M. Feig and Z. Burton.                  Role	of	Proline	in	Human	Fibroblast	Growth	Factor-1 L.
Michigan State Univ.                                                  Hall, L. Rutherford and T.K. S. Kumar. Univ. of Arkansas.
                                                                      B111     380      Probing Subunit Interactions in 3
                                                                      Phosphoglycerate Dehydrogenase B. Falk and E. Bell.
Protein Quality Control
B100 369           Use of a Laser-Scatter Triggering                  Univ. of Richmond.
Mechanism	 for	 the	 Imaging	 and	 Accurate	 Counting	 of	            B112 381	         Crystal	 Structure	 of	 the	 APOBEC3G	
Sub-visible Particles in Sparse Protein Samples L. Brown.             Catalytic	Domain	and	Functional	Implications	of	Putative	
Fluid Imaging Technol. Inc., Yarmouth, ME.                            Intermolecular Interfaces S. Shandilya, M. Nalam, P.
B101 370	          Mutagenesis	 Reveals	 the	 Complex	                Gross, E. Harjes, H. Matsuo, R. Harris, M. Somasundaran
Relationships	 between	ATPase	 Rate	 and	 the	 Chaperone	             and C. Schiffer. Univ. of Massachusetts Med. Sch. and Univ.
Activities of E. coli Hsp70 (DnaK) L. Chang, A. Thompson,             of Minnesota, Minneapolis.
P. Ung, H. Carlson and J. Gestwicki. Univ. of Michigan.               B113 382          Structure and Function of the Alternative
                                                                      Complex	 III	 in	 the	 Photosynthetic	 Electron	 Transfer	
                                                                      Chain	of	Chloroflexus aurantiacus X. Gao, Y. Xin, J. Wen,
Protein Structure Prediction                                          P. Bell and R. Blankenship. Washington Univ. in St. Louis.
                                                                      B114 383          Investigating Shape and Mechanism of
B102 371          Protein Domain Assignment from the
                                                                      Soluble	Guanylate	Cyclase B. Fritz, S. Roberts, J. Brailey,
Recurrence of Locally Similar Structures C-H. Tai, V.
                                                                      R. Purohit and W. Montfort. Univ. of Arizona.
Sam, J-F. Gibrat, J. Garnier, P. Munson and B. Lee. NCI and
                                                                      B115 384	         Model	for	Truncated	Soluble	Guanylate	
CIT, NIH and INRA, Jouy-en-Josas, France.
                                                                      Cyclase	by	negative	Stain	Electron	Microscopy J. Lemon,
B103 372	         Context	 Code	 of	 Protein	 Folding M.
                                                                      J. Brailey, S. Roberts and W. Montfort. Univ. of Arizona.
Apostol and I. Apostol. UCLA and Amgen, Thousand Oaks.
                                                                      B116 385	         The	 Effect	 of	 Distal	 Heme	 Pocket	
B104 373          Toward Understanding the Mechanism
                                                                      Mutations on the Water Accessible Areas in Myoglobin
of Action of the Yeast Multidrug Resistance Transporter
                                                                      B. Rodriguez, R. Goldbeck, R. Esquerra, R. Nguyen, D.
Pdr5p: A Molecular Modeling Study R.M. Rutledge, L.
                                                                      Kliger and A. Guliaev. San Francisco State Univ. and Univ.
Esser, J. Ma and D. Xia. NCI, NIH.
                                                                      of California, Santa Cruz.
B105 374          NMR Structure Determination by
                                                                      B117 386          Role of Proline Residues in the
Conformational	Space	Annealing J. Lee, J. Lee, W.T. Lee,
                                                                      Structure and Activity of the Human Acidic Fibroblast
B. Brooks and J. Lee. Korea Inst. for Adv. Study, Seoul,
                                                                      Growth	Factor-1 L. Rutherford and T.K. S. Kumar. Univ. of
Kwangwoon Univ. and Yonsei Univ., South Korea and NIH.

                                       TUESDAy,	AUGUST	3
B118 387	          The	 Dimerization	 of	 the	 Cytosolic	           B130 399          Examining the Structural Basis of
Domain of Fis1 May Regulate Mitochondrial Fission in                Ubiquitin Ligase Self-Assembly and Its Implication as a
Yeast J. Lees, L. Picton, C.M. Manlandro, S. Casares Atienza        Regulatory Switch W. Errington, A. Ross and G. Privé. Univ.
and B. Hill. Johns Hopkins Univ.                                    of Toronto and Ontario Cancer Inst., Univ. Hlth. Network,
B119 388	          Exploring	 Conformational	 Changes	              Toronto.
Associated	 with	 Binding	 of	 yC-1	 to	 Soluble	 Guanylyl	         B131 400          Exploring the Functional Landscape of
Cyclase M. Davis, C. Aguilera, S. Cooney and M. Gage.               the	Expanding	Interleukin-1	Family K. Hailey, K. Fisher
Northern Arizona Univ.                                              and P. Jennings. UCSD.
B120 389           Structural Investigations of Soluble             B132 401          Structure-Function Analysis of an
Oligomers of a-Synuclein I. Perovic, Q. Hoang, D. Ringe,            Archaeal	Type	Phosphoenolpyruvate	Carboxylase	Reveals	
G. Petsko and T. Pochapsky. Brandeis Univ. and Indiana              a New Inhibition Mode for Aspartate L. Dharmarajan, J.
Univ. Sch. of Med.                                                  Kraszewski, B. Mukhopadhyay and P. Dunten. Virginia Tech,
B121 390           Pro-IL-1b	 Shares	 a	 Core	 Region	              Virginia Bioinformat. Inst. and Stanford Univ.
of	 Stability	 as	 Compared	 with	 Mature	 IL-1b While              B133 402          Homodimeric Hapten Recognition by a
Maintaining	 a	 Distinctly	 Different	 Configurational	             Heavy-Chain	Only	Camelid	Antibody G. Sonneson and J.
Landscape: A Hydrogen/Deuterium Exchange Mass                       Horn. Northern Illinois Univ.
Spectrometry Study K. Hailey, S. Li, M. Andersen, M. Roy,
V. Woods, Jr. and P. Jennings. Sch. of Med., UCSD and Novo
Nordisk, Malov, Denmark.
                                                                    Protein Therapeutics and
B122 391	          Crystal	 Structure	 of	 Escherichia coli         Targets
NADPH FMN Reductase SsuE with and without Bound
FMN C. Driggers, H. Ellis and P.A. Karplus. Oregon State            B134 403	          Targeting	to	Rescue	p53	Cancer	Mutants	
Univ. and Auburn Univ.                                              with Small Molecule Drugs O. Demir, F. Salehi, C.D.
B123 392           Molecular Functional Analysis of                 Wassman, R. Baronio, G.W. Hatfield, R.E. Amaro, P. Kaiser
TTHB178 from Thermus thermophilus HB8 A. Shimada,                   and R.H. Lathrop. Univ. of California, Irvine.
K. Fukui, N. Nakagawa, R. Masui and S. Kuramitsu. Osaka             B135 404	          Engineered	 Bispecific	 Proteins	 That	
Univ. and RIKEN Spring-8 Ctr., Hyogo, Japan.                        Simultaneously	 Bind	 to	 and	 Inhibit	 Both	 VEGFR2	 and	
B124 393	          RSK1	 Has	 a	 Conserved	 Regulatory	             Alphav	Beta3	Integrin	for	Cancer	Therapy	and	Diagnosis
Element That Is Essential for Its Interaction with PKA R.           J. Cochran, A. Silverman and N. Papo. Stanford Univ.
Ilouz, A. Kornev and S. Taylor. UCSD and HHMI.                      B136 405           Tools and Techniques for Discovery of
B125 394           Dynamic         Differences      between         Adnectins,	a	novel	Class	of	Targeted	Biologics S. Low, M.
Structurally	 Conserved	 Bacterial	 and	 Human	                     Dabritz, S. Fong, L. Truechek and M. Gosselin. Adnexus, a
Dihydrofolate Reductases G. Bhabha, H.J. Dyson and P.E.             BMS R&D Co., Waltham, MA.
Wright. The Scripps Res. Inst.                                      B137 406	          A	 Unique	 Characterization	 Strategy	
B126 395	          Crystal	 Structure	 of	 an	 Archaeal	            for	a	Monoclonal	Antibody	DOTA	Conjugate	Utilizing	a	
Cleavage	and	Polyadenylation	Specificity	Factor	Subunit	            Maleimide	Linkage L. Jones, Y. Zhang, L. Taylor, D. Plan,
from Pyrococcus horikoshii Y. Nishida, H. Ishikawa, N.              B. Affholter, A. Senczuk, A. Hewig and R. Bailey. Amgen,
Nakagawa, S. Kuramitsu and R. Masui. Osaka Univ. and                Seattle.
RIKEN SPring-8 Ctr., Hyogo, Japan.                                  B138 407           Selective Prodrug Activation in the
B127 396           Dissecting the pH-Dependent Enzymatic            Presence	 of	 the	 Tumor-Marker	 HIF-1a by Engineered
Mechanism	 of	 the	 Sialyltransferase	 CstII	 from	                 Protein Switches C. Wright, J. Eshleman and M. Ostermeier.
Campylobacter jejuni by NMR Spectroscopy P. Chan, L.L.              Johns Hopkins Univ. and Johns Hopkins Univ. Sch. of Med.
Lairson, H.J. Lee, W.W. Wakarchuk, N.C. J. Strynadka, S.G.          B139 408	          Characterization	 of	 Inhibitory	 Anti-
Withers and L.P. McIntosh. Univ. of British Columbia, The           IGF-1R	Antibodies	with	Different	Epitope	Specificity	and	
Scripps Res. Inst. and Natl. Res. Council of Canada, Ottawa.        Ligand	 Blocking	 Properties	 Using	 Isothermal	 Titration	
B128 397           A Possible Role for Small Insertion and          Calorimetry	and	Surface	Plasmon	Resonance M. Beach,
Deletion	Events	in	the	Evolution	of	Cro	Protein	Folds K.V.          V. Frasca and S. Demarest. GE Healthcare, Northampton,
Eaton, M.R. Nelson and M.H. J. Cordes. Univ. of Arizona.            MA and Biogen Idec, San Diego.
B129 398           Inhibition of the S100B-p53 Interaction          B140 409           RLIP76: A Wide Spectrum Target for
Using Small Molecules Derived from Pentamidine L.                   Cancer	 Therapeutics S. Singhal, S. Yadav, J. Singhal, Y.
Thompson, S. Kudrimoti, P. Wilder, A. Coop, A. MacKerell            Awasthi and S. Awasthi. Univ. of North Texas Hlth. Sci. Ctr.
and D. Weber. Univ. of Maryland Baltimore.

                                        TUESDAy,	AUGUST	3
B141 410          Sesame Extract Reduces Brain Edema                 B153 422	         Recognition	 of	 Influenza	 Virus	
by Regulating Aquaporin 4 Protein Expression Not by                  Hemagglutinin by Neutralizing Antibodies D. Ekiert, R.
Inhibiting	Blood	Brain	Barrier	Damage	in	Stroke	Animal	              Xu and I.A. Wilson. The Scripps Res. Inst.
Model Y. Bu, W.M. Yang, S.K. Park, M.S. Chang, S.H. Park,            B154 423          Small Peptide Molecules’ Effects on
K.S. Kim, B.J. Kim, D. Lee and H. Kim. Col. of Oriental              Cancer	 Cells J. Goswami, A. Alileche and G. Hampikian.
Med., Kyung Hee Univ. and Mokpo Natl. Univ., South                   Boise State Univ.
B142 411          Screening for Protease Inhibitors of
Two Viral Systems and Reducing Agent Effect on Hit
                                                                     Protein-Protein and Protein-
Compounds H. Lee, R. Chaudhuri, A. Mittal, J. Torres and             Ligand Interactions
M. Johnson. Univ. of Illinois at Chicago.
B143 412	         Design,	 Expression	 and	 Purification	 of	        B155 424          Probing the Subunit Interface in
Trojan Horse Inhibitor-1, a Potential Protein Therapeutic            Glyoxysomal	 Malate	 Dehydrogenase E. Bell. Univ. of
against HIV J. Osterhout, K. Louie and K. Fryar. Angelo              Richmond.
State Univ.                                                          B156 425          Mapping the Interactions of AdV5 and
B144 413          Memory         Enhancing     Effect     of         AdV12	 E1A	 CREB-Binding	 Protein	 and	 TATA-Binding	
Yeunjachongmyungtang Extract on Scopolamine-                         Protein P. Haberz, M. Yamout, H.J. Dyson and P.E. Wright.
Induced	Dementia	Rats	through	the	Induction	of	Choline	              The Scripps Res. Inst.
Acetyltransferase Protein Expression D.R. Kim, Y.M. Bu,              B157 426          The Domains of Thrombomodulin and
W.M. Yang, S.K. Park and M.S. Chang. Col. of Oriental                Their	Ability	to	Alter	the	Catalytic	Activity	of	Thrombin
Med., Kyung Hee Univ., South Korea.                                  N. Treuheit and E. Komives. UCSD.
B145 414          Protective Effects of Palmijihwangwhan             B158 427          The Role of Histidines in Neurosteroid
against Oxidative Stress by Increasing Antioxidant                   Binding of NMDA NR2B and D Subunits S. Rhoads, L.
Enzymes	and	Activities	on	GC-1	spg	Male	Reproductive	                Zemcik and L. Gentile. Univ. of Richmond.
Cells E. Ko, W.M. Yang, S.K. Park and M.S. Chang. Col. of            B159 428          FtsA Assembles into Dynamic Polymers
Oriental Med., Kyung Hee Univ., South Korea.                         and	Enhances	FtsZ	Polymerization P. Singh, R. Makde, V.
B146 415          The Antioxidative Enzyme Activity of               Kumar and D. Panda. Indian Inst. of Technol. Bombay and
Woogyuyeum	 Extract	 on	 Leydig	 TM3	 Cells K. Chung,                Bhabha Atomic Res. Ctr., Mumbai.
H.Y. Kim, W.M. Yang, M.S. Chang and S.K. Park. Col. of               B160 429          Prediction of Substrates for Various
Oriental Med., Kyung Hee Univ., South Korea.                         Cytochromes	 P450	 Using	 a	 new	 Method S. Lee and D.
B147 416	         Effects	 of	 Cinnamomi	 Cortex	 on	                Kim. KAIST, Daejeon, South Korea.
Antioxidant Enzymes Activities, Androgen Activity in                 B161 430	         Using	 a	 Combination	 of	 FFT	 Docking	
Mouse	TM3	Leydig	Cells J. Yoon, B.J. Choi, W.M. Yang,                and Rigid-Body Minimization to Screen Amantadine-
M.S. Chang and S.K. Park. Col. of Oriental Med., Kyung               Like	Compounds	for	M2	Inhibition B. Hannigan, C. Soto,
Hee Univ., Korea and Republic of (South).                            J. Wang and W. DeGrado. Univ. of Pennsylvania.
B148 417          Targeting Protein Kinases with a Bivalent          B162 431          Energetic Analysis of the Interaction
Inhibition Strategy K. Cox, C. Shomin and I. Ghosh. Univ.            between Immunoglobulin A and FcaRI M. Brooks and A.
of Arizona.                                                          Herr. Univ. of Cincinnati.
B149 418          Investigating Molecular Mechanisms of              B163 432          Peptide Binding to Mutants of a System
Specificity	in	Regulation	of	the	HER2	and	HER4	Protein	              Specific	Chaperone	Differentiates	Stability	from	Affinity
Kinases through Theory and Experiment S. Telesco, R.                 T. Winstone, T. Leach and R. Turner. Univ. of Calgary,
Vadigepalli and R. Radhakrishnan. Univ. of Pennsylvania              Canada.
and Thomas Jefferson Univ.                                           B164 433          Designing       Novel      Antimicrobial
B150 419	         Controlling	 Physicochemical	 Behavior	            Compounds	 to	 Inhibit	 Unexplored	 Metabolic	 Pathways
of	a	Polymeric	Anticancer	Therapeutic:	A	Coarse	Grained	             A. Allen, S. Gilmore, R. Molloy, A. Grace, J. McCabe, C.
Modeling Study L. Peng and D. Gough. UCSD.                           Browder and M. Gage. Northern Arizona Univ.
B151 420	         Characterization	 and	 Informed	 Design	           B165 434	         Identification	 of	 Compounds	 That	
of	 Downregulating	 Anti-epidermal	 Growth	 Factor	                  Inhibit Tryptophan Synthase Dimerization R. Molloy,
Receptor Antibodies J. Spangler and K.D. Wittrup. MIT.               A. Allen, J. McCabe, A. Grace, C. Browder and M. Gage.
B152 421	         SIRT1	Overexpression	Protects	Cardiac	             Northern Arizona Univ.
Cells	 Against	 Ischemia-Reperfusion	 Injury M. Becatti,
C. Fiorillo, N. Nassi, N. Taddei, C. Cecchi, G. Soldi and P.
Nassi. Univ. of Florence and Children’s Hosp., Florence.

                                       TUESDAy,	AUGUST	3
B166 435	       More	Than	Just	a	Tether?	the	Role	of	the	
Inter-domain	Linker	in	Regulating	AraC’s	in	Vivo	and	in	            Receptors, Signaling and
Vitro Behavior J. Seedorff and R. Schleif. Johns Hopkins
                                                                    Signal Transduction
B167 436        Understanding the Role of Rob’s                     B168 437	        HDAC3	 Selectively	 Represses	 CREB3-
C-Terminal	 Domain	 in	 the	 Sequestration	 Dispersal	              Mediated	 CXCR4	 Expression	 and	 Migration	 of	 MDA-
Mechanism of Induction in E. coli L. Taliaferro, N. Sanchez-        MB-231	 Metastatic	 Breast	 Cancer	 Cells K-C. Choi and
Alberola and R.E. Wolf, Jr. Univ. of Maryland Baltimore             H-G. Yoon. Yonsei Univ. Col. of Med., South Korea.
County.                                                             B169 438	        Tissue	 Specific	 Interactions	 of	 Plasma	
                                                                    Membrane Proteins as the Mechanism of Functional
                                                                    Diversity J. Hwang, Y-E. Shin, J. Kim, S. Park and S. Kim.
                                                                    POSTECH, South Korea.
                                                                    B170 439         Molecular Signaling Mechanism of the
                                                                    Temperature-Sensing Histidine Kinase DesK J. Forman,
                                                                    C. Merlet and M. Nilges. Inst. Pasteur, Paris.

                       POSTER SESSION 3
                 Wednesday, August 4, 2010
           Exhibit Hall/Douglas Pavilion, 1st Level

                                       Poster Set Up Time
                                       7:00 AM – 8:00 AM

                                      Poster Viewing Time
                                       8:00 AM – 3:15 PM

                               Author Presentations
                   Odd Board Numbers                                          1:45 PM – 2:30 PM
                   Even Board Numbers                                         2:30 PM – 3:15 PM

                                Poster Removal
                               3:15 PM – 3:30 PM
        IMPORTANT: The Exhibit Hall will close promptly at 3:30 PM.
    Please remove posters by 3:30 PM. We are not responsible for lost posters.

Sessions                                                                                            Board Numbers
Amyloids and Protein Misfolding............................................................B1 – B21
Bioinformatics, Genomics, Proteomics ...................................................B22 – B30
Chromatography and Protein Purification ...............................................B31 – B39
Computational Biology ............................................................................B40 – B48
Enzyme Kinetics and Mechanism ............................................................B49 – B52
Membrane Proteins ..................................................................................B53 – B68
New and Developing Methods.................................................................B69 – B83
Protein Design and Protein Engineering ..................................................B84 – B103
Protein Dynamics: Theory and Experiment .............................................B104 – B109
Protein Folding: General Aspects ............................................................B110 – B124
Protein Formulation and Process Development.......................................B125 – B129
Protein Structure/Function Studies ..........................................................B130 – B170

                                     WEDnESDAy,	AUGUST	4
                                                                     B14       453	     A	 Biophysical	 Method	 to	 Specifically	
Amyloids and Protein                                                 Detect Ab Soluble Oligomers J. Finke, J. Digambaranath,
                                                                     D. Camp, T. Campbell, R. Colby, L. Dang, B. House, A.
Misfolding                                                           Klaver, L. Patrias, C. Walby and D. Loeffler. Oakland Univ.
B1        440     Development of a Beta-Hairpin Model                and Beaumont Hosp. Res. Inst., Royal Oak, MI.
to	 Study	 the	 Role	 of	 PolyQ	 Repeats	 in	 Cross-Beta-            B15       454      Molecular Tweezers as Broad-Spectrum
Sheet Aggregation. A. Cuesta, B. Kokona and R. Fairman.              Inhibitor of Amyloid Protein Aggregation and Toxicity D.
Haverford Col.                                                       Lopes, S. Sinha, A. Tennstädt, M. Ehrmann, F-G. Klärner,
B2        441     Amyloid Fibrils Are Alive: Spontaneous             T. Schrader and G. Bitan. UCLA and Univ. Duisburg-Essen,
Refolding from One Polymorph to Another D. Kurouski,                 Germany.
W. Lauro and I. Lednev. Univ. at Albany, SUNY.                       B16       455      Insights on Aggregation of Alpha-
B3        442     Mechanistic Insights into Unfolding of             Synuclein: Simulations on Fibril Forming Peptides and
Amyloid	Beta-Peptide	Central	Helix M. Ito, J. Johansson,             the Acidic Domain S. Shin, S.B. Park, J. Yoon, S. Jang and
R. Stromberg and L. Nilsson. Karolinska Inst., Huddinge and          K. Lee. Seoul Natl. Univ. and Sejong Univ., South Korea.
Swedish Univ. of Agr. Sci.                                           B17       456      Novel Tools to Further the Study of
B4        443	    Site	 Specific	 Fluorescent	 Probes	 of	           Light	Chain	Amyloidosis L. Ryno, J. Hulleman, W. Balch
a-Synuclein Fibril Assembly T. Yap, C. Pfefferkorn and J.            and J. Kelly. The Scripps Res. Inst.
Lee. NHLBI, NIH.                                                     B18       457      Functional Amyloids as Hormones
B5        444     Amyloid Formation Kinetics and                     Storage	in	Pituitary	Secretory	Granules S. Maji, A. Anoop,
Fibrillar Structures of the Essential Melanosomal Protein            R. Jacob, P. Singru and R. Riek. Indian Inst. of Technol.,
Pmel17 J. Lee, C. Pfefferkorn, R. McGlinchey and R.                  Bombay, Tufts Med. Ctr. and ETH Zurich.
Wickner. NHLBI and NIDDK, NIH.                                       B19       458      Examining       the     Mechanism        of
B6        445     Study of Early Events in Ab Aggregation            Glycosaminoglycan-Accelerated	 Amyloidogenesis J.
by EPR Spectroscopy A. Agopian, S. Ngo and Z. Guo.                   Solomon and J. Kelly. The Scripps Res. Inst.
UCLA.                                                                B20       459      Amyloid       Aggregation        Mitigating
B7        446     Mechanistic          Investigation       of        Peptides C. Bett, W. Serem, J. Garno and R. Hammer. UCSD,
Tetrapeptide Inhibitors of Ab42-Induced Neurotoxicity                LSU and New England Peptide, Gardner, MA.
H. Li, E. Fradinger and G. Bitan. UCLA.                              B21       460	     Copper	II	and	H2O2 Mediated b-Cleavage	
B8        447     Isolation of Short Peptide Segments                of Recombinant Prion Protein A. McDonald, A. Lau, R.
from	 Alpha-Synuclein	 Fibril	 Core	 Identifies	 a	 Residue	         Aglietti, E. Walter, D. Westaway and G. Millhauser. Univ. of
Important for Fibril Nucleation Y. Kawata, H. Yagi, H.               California, Santa Cruz, Univ. of Alberta and Pacific Northwest
Takeuchi, K. Hongo, T. Mizobata and Y. Goto. Tottori Univ.,          Natl. Lab., Richland, WA.
Japan and Osaka Univ.
B9        448     Folding      Stability      and
Conformation	of	Amyloid-Beta	Monomers	Are	Important	
                                                      Native         Bioinformatics, Genomics,
Determinants of Nucleation Kinetics and Fibril Formation             Proteomics
Y-R. Chen and C-L. Ni. Genomics Res. Ctr., Acad. Sinica,
                                                                     B22      461     Structural and Functional Analysis of
                                                                     NS1 and NS2 Proteins of H1N1 Subtype P. Salahuddin.
B10       449     Novel Inhibitors of IAPP Assembly and
                                                                     Aligarh Muslim Univ., India.
Cytotoxicity D. Lopes, K.A. McDaniel, T. Schrader, F-G.
                                                                     B23      462	    A	 Short	 Survey	 on	 Protein	 Blocks
Klärner and G. Bitan. UCLA and Univ. Duisburg-Essen,
                                                                     A. de Brevern, A.P. Joseph, G. Agarwal, N. Srinivasan
                                                                     and C. Etchebest. INSERM, Paris and Indian Inst. of Sci.,
B11       450	    The	 Influence	 of	 Zn2+	 on	 the	 Global	
Structure of the Prion Protein A. Spevacek, A. Howard,
                                                                     B24      463     Detecting Internally Symmetric Proteins
J. Pelton and G. Millhauser. Univ. of California, Santa Cruz,
                                                                     in the Protein Domain Structure Database B. Lee, C. Kim
Cabrillo Col. and Univ. of California, Berkeley.
                                                                     and J. Basner. NCI, NIH and Yonsei Univ., South Korea.
B12       451	    Characterisation	 of	 the	 in	 Vivo	
                                                                     B25      464     Using the PSI Structural Biology
Misfolding Behaviour of the Lysozyme Amyloid Variant
                                                                     Knowledgebase to Predict Novel Annotations for Under-
F57I Using Drosophila melanogaster L. Andrén and A-C.
                                                                     Characterized	 Proteins W. McLaughlin, E. Julfayev, R.
Brorsson. Linkpöing Univ., Sweden.
                                                                     McLaughlin, J. Westbrook, W.Y-P. Tao, R. Shah, M. Gabanyi
B13       452	    In	 Vitro	 Characterization	 of	 the	
                                                                     and H. Berman. Commonwealth Med. Col., PA and Rutgers
Aggregation Process for Amyloid Beta 42 Variants That
                                                                     Univ., Piscataway.
Differ in Their in Vivo Toxicity A-L. Göransson and A-C.
Brorsson. Linkpöing Univ., Sweden.

                                   WEDnESDAy,	AUGUST	4
B26       465     NMR Data for Proteins at BMRB:                    B37      476	     Tips	and	Hints	for	Purification	of	GST-
Expanding	 Data	 Types	 and	 new	 Query	 Capabilities H.            Tagged	Proteins	Using	Prepacked	Columns A. Heijbel, L.
Yao, H. Akutsu, Y. Harano, Y. Ioannidis, N. Kobayashi, M.           Lilja and J. Lundqvist. GE Healthcare, Uppsala.
Livny, S. Mading, D. Maziuk, E. Nakatani, C. Schulte, E.            B38      477	     Automated	      Protein	      Purification:	
Ulrich, R.K. Wenger and J. Markley. Univ. of Wisconsin-             Reducing Delivery Time of High Quality Protein Reagents
Madison, Osaka Univ. and Univ. of Athens.                           from Days to Hours A. Varghese, T. Subashi, M. Griffor,
B27       466     Detection of Remote Similarities in               I-K. Wang, P. Loulakis and J. Culp. Pfizer Inc.
Protein	Structures	in	the	RCSB	PDB A. Prlic, B. Beran, C.           B39      478	     Expression	 and	 Purification	 of	
Bi, S. Bliven, W.F. Bluhm, D. Dimitropoulos, G.B. Quinn, B.         Arabidopsis	CRInKLy4	(ACR4)	Subdomains	and	ACR4	
Yukich, P.E. Bourne and P.W. Rose. Skaggs Sch. of Pharm.            Related Proteins A.G. Rao and M. Meyer. Iowa State Univ.
and Pharmaceut. Sci., UCSD.
B28       467     The Power of a Perfect Alignment:
Correlating	Co-evolution	of	Species,	Cofactor	and	Enzyme	
                                                                    Computational Biology
Function W. Duax, R. Huether and D. Dziak. Hauptman-                B40       479       Drugable Exosites of the Human Kino-
Woodward Inst., Buffalo.                                            pocketome G. Nicola, I. Kufareva, F. Bauchinger and R.
B29       468     Bioinformatic Search for Protease                 Abagyan. UCSD.
Substrates J. Johnson, E. Chambers and K. Jayasundera.              B41       480       Predicting Ligand Binding Sites in
Univ. of Minnesota Duluth.                                          Protein	Complexes	and	Allosteric	Enzymes S. Mitternacht
B30       469	    Getting	 the	 Most	 Out	 of	 the	 RCSB	           and I. Berezovsky. Univ. of Bergen, Norway.
Protein	Data	Bank A. Prlic, B. Beran, C. Bi, S. Bliven, W.F.        B42       481       Dimensionality Reduction Techniques
Bluhm, D. Dimitropoulos, G.B. Quinn, B. Yukich, P.E. Bourne         for	the	Study	of	Protein	Conformations L. Han, M. Duan
and P.W. Rose. Skaggs Sch. of Pharm. and Pharmaceut. Sci.,          and S. Huo. Clark Univ.
UCSD.                                                               B43       482       Development of the Molecular Software
                                                                    Libraries for Modeling and Designing Proteins D. Kulp,
Chromatography and Protein                                          B. Hannigan, J. Donald, S. Subramaniam, B. Mueller and
                                                                    A. Senes. Los Alamos Natl. Lab., Univ. of Pennsylvania and
Purification                                                        Univ. of Wisconsin-Madison.
                                                                    B44       483	      Exploring	 Protein	 Conformational	
B31      470	     Purification	 of	 MBP-Tagged	 Proteins	           Changes	with	Accelerated		Molecular	Dynamics	in	nAMD
Using	Prepacked	Columns A. Heijbel, A. Bergh, J. Zou, A.            Y. Wang and J.A. McCammon. HHMI, UCSD.
Karlsson, J. Lundqvist, K. Busson, M. Carlsson, E. Maier and        B45       484       Protein Function Annotation by
R. Zimmerman. GE Healthcare, Uppsala, Dr. von Haunersches           Structurally Aligned Local Sites of Activity M. Ondrechen,
Kinderspital, Munich and Leiden Univ., Netherlands.                 J.S. Lee, P. Yin and S. Somarowthu. Northeastern Univ.
B32      471      Recombinant         Myostatin      Latent         B46       485       Structural      and        Thermodynamic
Complex	 Purification	 and	 Reconstitution H. Zhang, J.             Evidence for Allosteric Relaxed-State Symmetry R.
Trill, K. Johnson, S. Gillbert, F. Kull and K. Johanson.            Ettrich, R. Strawn, M. Melichercik, T. Stockner and J. Carey.
GlaxoSmithKline.                                                    Inst. of Syst. Biol. and Ecol., Acad. of Sci. of Czech Republic,
B33      472      A Method for the Prevention of                    Univ. of South Bohemia, Czech Republic, Princeton Univ.
Thrombin-Induced Degradation of Recombinant Proteins                and Med. Univ. of Vienna.
P. Adams, D. Rajalingam, K. Kathir, K. Ananthamurthy and            B47       486	      Polarizable	Protein	Packing C. Snow and
T.K. S. Kumar. Univ. of Arkansas.                                   A. Ng. Caltech.
B34      473      Monoclonal Antibody Heterogeneity                 B48       487	      A	Computational	Analysis	of	Molecular	
Characterization	Using	Cation	Exchange	Columns S. Rao,              Recognition in the Trypsin/BPTI System Using Multiple
Y. Hou, Y. Agroskin and C. Pohl. Dionex Corp., Sunnyvale,           Electrostatic Models M. Minkara, J.P. Bardhan and M.
CA.                                                                 Radhakrishnan. Wellesley Col. and Rush Univ. Med. Ctr.
B35      474	     High	 Capacity	 Small-Scale	 Antibody	
Purification	Using	Magnetic	Beads S. Eriksson, A. Bergh,
G. Glad, T. Graner, A. Grönberg, U. Hellberg and H. Hedlund.
GE Healthcare Life Sci., Uppsala.
B36      475	     Small-Scale	 Purification	 of	 Histidine-
Tagged Recombinant Proteins Using Magnetic Beads S.
Eriksson, A. Bergh, F. Borg, G. Glad, T. Graner, H. Hedlund,
U. Hellberg and J. Öhman. GE Healthcare Life Sci.,

                                    WEDnESDAy,	AUGUST	4
                                                                    B60       499	     Repression	 of	 Glucose	 Inducible	 Outer	
Enzyme Kinetics and                                                 Membrane Protein during Preferential Utilization of
                                                                    Aromatic	 Compounds	 Over	 Glucose	 in	 Pseudomonas
Mechanism                                                           putida	 CSV86 R. Shrivastava and P. Phale. Indian Inst. of
B49     488	      Characterization	 of	 Cobalt-Substituted	         Technol. Bombay, Mumbai.
PSAO S. Mills, K. Dang and J. Nguyen. Univ. of San                  B61       500	     Formation	 of	 Immunogenic	 Influenza	
Diego.                                                              Virus-Like	 Particles	 by	 Hemagglutinin	 Alone S-y. Lai,
B50     489	      Thermal	 Behaviour	 of	 Contemporary,	            G.R-L. Chang and M-Y. Wang. Central Taiwan Univ. of Sci.
Resurrected Ancestral and Mutant 3-Isopropylmalate                  and Technol. and Natl. Chung Hsing Univ., Taiwan.
Dehydrogenases J. Hobbs, C. Shepherd, D. Saul, R. Daniel            B62       501      The Membrane ERAD E3 Ligase Doa10
and V. Arcus. Univ. of Waikato, Univ. of Otago and ZyGEM            Is Split in Two Fragments in Kluyveromyces lactis S. Kreft,
Corp., Hamilton, New Zealand.                                       E. Stuerner, M. Scheffner and M. Hochstrasser. Univ. of
B51     490       ApoE Induces Serum Paraoxonase1                   Konstanz, Germany and Yale Univ.
Activity and Stability Similar to ApoA-I V. Nair, L.                B63       502      Towards the Prediction of All
Gaidukov, S. Yacobson, M. Rosenblat, M. Aviram and D.               Physiologically	 Important	 Conformations	 of	 a	 GPCR
Tawfik. Weizmann Inst. of Sci., Rehovot, MIT and Rambam             R. Abrol, J. Bray and W. Goddard III. Caltech and Stanford
Med. Ctr., Haifa.                                                   Univ.
B52     491	      Biochemical	 Characterization	 and	               B64       503	     CCR5:	 Structure	 Prediction,	 Ligand	
Mutational Analysis of Prephenate Dehydrogenase from                Docking,	and	Mutagenesis	Validation	Aimed	at	Disrupting	
Saccharomyces cerevisiae	and	Its	Seed-Specific	Expression	          HIV Entry W. Goddard III, B. Trzaskowski and R. Abrol.
for	 Increased	 Tocopherol	 Content	 in	 Transgenic	                Caltech.
Arabidopsis Seeds Q. Qi, M. Hao, S. Falker and H. Valentin.         B65       504	     A	 Combined	 Spectroscopic	 and	
Monsanto, St. Louis, Ballwin, MO and Davis, CA.                     Biochemical	Approach	for	Counting	Channel	Subunits T.
                                                                    Walton, C. Gandhi and D. Rees. Caltech and HHMI, Chevy
                                                                    Chase, MD.
Membrane Proteins                                                   B66       505      Statistical Potentials Enable Improved
B53      492      Regulation of M. tuberculosis	 Cell	              Modeling of Membrane Proteins D. Kulp, J. Donald and W.
Division:	 Discovery	 of	 Protein	 Complexes N. Das, C.             DeGrado. Los Alamos Natl. Lab. and Univ. of Pennsylvania.
Avalos and T.A. Cross. Florida State Univ. and Natl. High           B67       506	     novel	 Inhibitors	 of	 CLC	 Chloride	
Magnetic Field Lab.                                                 Channels	and	Transporters M. Maduke, A. Howery, J. Du
B54      493      How prl Mutations or Binding of the               Bois, S. Philips and K. Matulef. Stanford Univ. and Univ. of
Signal	Peptide	Unlock	the	Protein	Translocon A.N. Bondar,           San Diego.
D.J. Tobias and S.H. White. Univ. of California, Irvine.            B68       507      Structure, Dynamics, and Drug
B55      494	     Dimerization	of	Gram-Positive	Bacterial	          Interactions of the p7 Membrane Protein from Hepatitis
Membrane Transpeptidase Sortase A Has Big Impact on                 C	Virus	by	nMR G. Cook, Y. Mai and S. Opella. UCSD.
Its Activity in Vivo Z. Zhang and J. Zhu. Univ. of Texas at
Austin.                                                             New and Developing Methods
B56      495      Oxygen Regulated Protein Degradation
of	the	Outer	Membrane	Decaheme	Cytochrome	MtrC	in	                  B69      508     Pharmacophore Modeling Strategies
the Bacterium Shewanella oneidensis MR-1 Y. Xiong, L.               for the Development of Novel Nonsteroidal Inhibitors of
Shi, D. Bigelow and T. Squier. Pacific Northwest Natl. Lab.,        Human	Aromatase	(CyP19) Y. Muftuoglu and G. Mustata.
Richland, WA.                                                       Johns Hopkins Univ. and Univ. of Pittsburgh Sch. of Med.
B57      496	     The	       Transmembrane	            CadC	        B70      509	    new	Methods	for	Cleavable	Modification	
Transcriptional Activator of the Acid-Response cadBA                of	Cysteine-Containing	Peptides	and	Molecular	Imaging	
Operon Is a Peripheral Membrane Protein E. Lindner and              of	Cysteine	and	Homocysteine M.K. Wong, H.Y. Shiu and
S.H. White. Univ. of California, Irvine.                            C.M. Che. The Hong Kong Polytech Univ. and The Univ. of
B58      497      Transmembrane                Helix-Driven         Hong Kong.
Dimerization of Single-Pass Receptors R. Worch, T.                  B71      510	    Development	of	Gold	Catalysis	for	Site-
Weidemann, C. Boekel and P. Schwille. Tech Univ. Dresden,           Selective	 Modification	 of	 Glycoproteins K.Y. K. Kung,
Germany.                                                            C.M. Che and M.K. Wong. The Hong Kong Polytech Univ.
B59      498	     Protein	 Clustering	 within	 a	 Lipid	            and The Univ. of Hong Kong.
Membrane A. Hamill, L. Porcar and P. Butler. NIST,
Gaithersburg, MD and Inst. Laue-Langevin, Grenoble.

                                    WEDnESDAy,	AUGUST	4
B72      511      High Frequency Micro-rheological                   B83     522     Long-Term Stabilization of Functional
Characterization	 of	 Protein	 Solutions	 Using	 QCM-D               Bacterial	Photosynthetic	Reaction	Centers	in	Substrate-
M. Dixon, A. Oom, C. Cheng, M. Johal and C. Cheney.                  Supported Lipid Nanotube Arrays A. Smirnov and O.
BiolinScientific, Glen Burnie, Q-Sense, Gothenburg and               Poluektov. North Carolina State Univ. and Argonne Natl.
Pomona Col.                                                          Lab.
B73      512	     An	 Efficient,	 Integrated	 Platform	 for	
Protein Expression in both Spodoptera and Mammalian
Cell	 Systems W. Hou, T. Ho and R. Kirkpatrick.
                                                                     Protein Design and Protein
GlaxoSmithKline.                                                     Engineering
B74      513      High Resolution Mapping of Protein
Sequence-Function Relationships D. Fowler, C. Araya, S.              B84      523	     Expression,	       Cross-Linking	     and	
Fleishman, E. Kellogg, J. Stephany, D. Baker and S. Fields.          Characterization	 of	 Recombinant	 Resilin	 with	 Chitin	
Univ. of Washington and HHMI, Chevy Chase, MD.                       Binding G. Qin, S. Lapidot, K. Numata, X. Hu, S. Meirovitch,
B75      514	     Expresso™	T7	Cloning	and	Expression	               M. Dekel, I. Podoler, O. Shoseyov and D. Kaplan. Tufts Univ.
System D. Mead, R. Godiska, V. Dhodda, L. Sheets, C.                 and Hebrew Univ. of Jerusalem.
Niebauer and E. Steinmetz. Lucigen, Middleton, WI.                   B85      524	     Interrogating	 Cellulase	 Function	 with	
B76      515	     Glycosylated	Human	Cardiothropin-1	Is	             Structure-Guided	Recombination M. Farrow, C. Snow, P.
Functional	and	Can	Be	Produced	at	High	Levels	in	Stable	             Romero and F. Arnold. Caltech.
Cells	with	a	Semliki	Forest	Virus	noncytopathic	Vector A.            B86      525	     Generation	 of	 Longer	 Emission	
Aranda, E. Casales, J.I. Quetglas, M. Ruiz-Guillen, J. Prieto        Wavelength       Red      Fluorescent     Proteins    Using
and C. Smerdou. Univ. of Navarra , Pamplona.                         Computationally	Designed	Libraries R. Chica, M. Moore,
B77      516	     Purification	 and	 Characterization	 of	           B. Allen and S.L. Mayo. Univ. of Ottawa and Caltech.
Biotinylated	Caspase-6 H. Li, M. Elliott, B. Brilliantes, K.         B87      526      Proteins from a de Novo Designed
Mortara, C. Tam, Y. Franke, K. Bowman, K. Shah and J. Wu.            Library Exhibit Selective Binding to a Range of Small
Genentech.                                                           Molecules I. Cherny, A.N. Koehler and M.H. Hecht. Princeton
B78      517	     Microvolume	Quantification	of	Proteins	            Univ. and Broad Inst. of Harvard and MIT.
by UV-Vis Absorbance or Fluorescence D. Ash and A.                   B88      527      Engineering Antigen Binding Sites
Page. Thermo Scientific NanoDrop Products, Wilmington,               into	 the	 Fc	 Region	 of	 IgG:	 Biochemical,	 Biophysical	
DE.                                                                  and	 Immunological	 Characterization F. Rueker and G.
B79      518	     In	 Vitro	 Genetic	 Reconstruction	 of	            Wozniak-Knopp. Boku Univ., Austria.
Bacterial	 Transcription	 Initiation	 by	 Coupled	 Synthesis	        B89      528      Salt Bridges in Protein Design:
and Detection of RNA Polymerase Holoenzyme S. Chong                  Geometrically	Specific	Interactions J. Donald, D. Kulp and
and H. Asahara. New England Biolabs.                                 W. DeGrado. Univ. of Pennsylvania and Los Alamos Natl.
B80      519      Recent Advances in the Utilization of the          Lab.
Wheat	Germ	Cell-Free	Expression	System S-i. Makino, E.               B90      529	     Recombinant	Expression	of	a	Backbone-
Beebe, A. Nozawa, P.S. Chae, R. Wrobel, L. Bergeman, R.              Cyclized	Mouse	a-Defensin A. Garcia, A. Ouellette and J.
Frederick, Y. Matsubara, S. Gellman, F. Vojtik, J. Markley,          Camarero. Univ. of Southern California.
C. Bingman, G. Phillips, J. Primm, B. Volkman and B. Fox.            B91      530      In Vivo Encapsulation of Nucleic Acids
Univ. of Wisconsin-Madison and Med. Col. of Wisconsin.               Using	 an	 Engineered	 Protein	 Capsid K. Woycechowsky
B81      520      Protein Domain Trapping Method:                    and S. Lilavivat. Univ. of Utah.
In	 Search	 of	 Compact,	 Soluble	 Protein	 Fragments	               B92      531      Design of Novel Binders for Ras,
for Structural Biology H.B. Nguyen, J-D. Pédelacq, S.                a	 Molecular	 Switch	 in	 Various	 Signaling	 Cascades
Cabantous, B. Mark, P. Listwan, C. Bell, N. Friedland,               J. Shifman, A. Erijman, Y. Peleg, D. Filchtinski and C.
T.C. Terwilliger and G.S. Waldo. CNRS, France, INSERM                Herrmann. Hebrew Univ. of Jerusalem, Weizmann Inst. of
U563, Département Innovation Thérapeutique et Oncologie              Sci., Rehovot and Ruhr Univ. Bochum, Germany.
Moléculaire, France, Los Alamos Natl. Laboratory and Univ.           B93      532	     Stabilizing	 the	 nKG2D-MICA	 Protein-
of Manitoba, Canada.                                                 Protein Interaction with Rational Design Both at and
B82      521      Dielectric and Hydrogen Bonding                    below the Interface B. McFarland, M. Peterson, J. Ban and
Gradients	 along	 an	 a-Helix in a Transmembrane                     K. Bickley. Seattle Pacific Univ.
Orientation T. Smirnova, M. Voynov and A. Smirnov. North             B94      533	     MD-Based	Calculation	of	Configuration	
Carolina State Univ.                                                 Integral Provides Thermodynamic Insight G. Grigoryan.
                                                                     Univ. of Pennsylvania.

                                     WEDnESDAy,	AUGUST	4
B95      534	      Computational	Enzyme	Design	–	Kemp	               B109 548	        Does	 a	 Mutation	 Predicted	 to	 Change	
Elimination Reaction M. Frushicheva, J. Cao and A.                   the	 Dynamics	 of	 Bacterial	 Phosphatidylinositol	 Specific	
Warshel. Univ. of Southern California.                               Phospholipase	C	Affect	Activity	and	Membrane	Binding?
B96      535       Rational Tuning of the Redox Potential            J. Cheng, S. Karri, M.F. Roberts, P.L. Wintrode and A.
of an Unusual FeS Protein J. Zuris, D. Halim, A. Conlan, E.          Gershenson. Boston Col., Case Western Reserve Univ. and
Abresch, R. Nechushtai, M. Paddock and P. Jennings. UCSD             Univ. of Massachusetts Amherst.
and The Hebrew Univ. of Jerusalem.
B97      536       Structural and Functional Studies
Shed	 Light	 on	 Increased	 HIV	 neutralization	 by	 Linked	
                                                                     Protein Folding: General
Cyanovirin-n	 Dimers J. Keeffe, P. Gnanapragasam, S.K.               Aspects
Gillespie, P.J. Bjorkman and S.L. Mayo. Caltech.
B98      537       Design of a Switchable Kemp Elimination           B110 549	          Influence	 of	 the	 Hydrophobic	 Core	 on	
Catalyst I. Korendovych, D. Kulp, Y. Wu, H. Cheng, H.                the Internal Friction of Folding: Experimental Studies of
Roder and W. DeGrado. Univ. of Pennsylvania and Fox                  the Four-Helix Bundle Protein Rop with Wild-Type and
Chase Cancer Ctr.                                                    Designed	 Cores C. Cheng, R. Ilagan, L.G. Kwa, J. Clarke
B99      538	      Chemical	 Modification	 of	 Trichoderma           and L. Regan. Yale Univ. and Univ. of Cambridge.
reesei	 Cel6A	 Improves	 Biomass	 Hydrolysis T. Kaper, A.            B111     550       Ligand-Dependence        Stability   and
Liu, L. Cascao-Pereira, S. Lantz, E. Larenas and B. Kelemen.         Folding	in	CusF,	a	Bacterial	Copper(I)	Chaperone S. Ly,
Genencor, a Danisco Div., Palo Alto.                                 D. Dally and B. Gillespie. California State Univ., Channel
B100 539           Oligonucleotide Recombination for                 Islands.
Affinity	Maturation	of	an	Fn3-Scaffold	Binding	Protein               B112 551           Differences in Folding Pathways of
M. Merguerian, J. Wojcik, A. Koide, S. Koide and V. Cornish.         Homologous Ribonucleases H. Scheraga, R. Gahl and R.
Columbia Univ. and Univ. of Chicago.                                 Oswald. Cornell Univ.
B101 540	          The	 Chemical	 and	 Synthetic	 Biology	           B113 552           NMR Analysis of a Kinetically Trapped
of a Man-Made Protein Evolved de Novo from Random                    Refolding	Intermediate	of	a	Disulfide-Deficient	Mutant	of	
Sequence Origin J. Chaput. Arizona State Univ.                       the	Starch-Binding	Domain	of	Glucoamylase H. Sugimoto,
B102 541           Interface Histidine-Scanning of a Protein         Y. Noda and S-i. Segawa. Grad. Sch. of Sci. and Technol.,
Interface to Modulate Protein-Protein Binding J. Horn,               Kwansei Gakuin Univ., Japan.
M. Murtaugh and S. Fanning. Northern Illinois Univ.                  B114 553	          Macromolecular	 Crowding	 of	 an	
B103 542           Multivalent Interactions with gp120               Unfolded Protein Studied by Small-Angle Neutron
Are	Required	for	the	Anti-HIV	Activity	of	Cyanovirin G.              Scattering. D. Johansen, B. Hammouda, J. Trewhella and
Ghirlanda, M. Ruben and B. Woodrum. Arizona State Univ.              D. Goldenberg. Univ. of Utah, NIST, Gaithersburg, MD and
                                                                     Univ. of Sydney.
                                                                     B115 554           Entamoeba histolytica	Cysteine	Protease	
Protein Dynamics: Theory and                                         Secondary Structure and Thermodynamic Stability
Experiment                                                           O. Zamudio, C. Benitez-Cardoza and J. Ortega-Lopez.
                                                                     CINVESTAV-IPN and ENMH-IPN, Mexico City.
B104 543           Finding Sodium Binding Sites in                   B116 555	          Chromatographic	        Refolding	     of	
Membrane Proteins: Na+/K+/ Binding and Selectivity in                Recombinant Hyaluronidase Assisted by Apical Domain
Sodium-Coupled	 Aspartate	 Transporter	 Glt S. Noskov                of	 GroEL,	 DsbA	 and	 DsbC	 Immobilized	 in	 Cellulose
and B. Lev. Univ. of Calgary, Canada.                                A. Antonio-Perez, H. Clement-Carretero, A. Alagon and J.
B105 544           NMR Relaxation Experiments for                    Ortega-Lopez. CINVESTAV-IPN, Mexico City and Natl.
Proteins at High-Magnetic Field Strength R. Ishima, W.               Autonomous Univ. of Mexico, Morelos.
Myint and L. Menon. Univ of Pittsburgh Sch. of Med.                  B117 556	          Bile	Acids	Can	Decrease	Enzyme	Activity	
B106 545	          Comparison	of	the	Dynamic	Properties	             by Modulating the Thermodynamic Stability of Proteins
of WT Protease and a Drug-Resistant Variant Y. Cai and               S. Robic, K. Linscott and M. Aseem. Agnes Scott Col., GA.
C. Schiffer. Univ. of Massachusetts Med. Sch.                        B118 557           Folding Study of Outer Surface Protein
B107 546           Allostery in Ras Signaling A. Gorfe. Univ.        A K. Makabe, T. Nakamura and K. Kuwajima. Okazaki Inst.
of Texas Hlth. Sci. Ctr. at Houston.                                 for Integrat. Biosci., Japan.
B108 547	          Determination	 of	 Backbone	 Dynamics	            B119 558           Structure and Mechanism of the
of	the	C2A	Domain	of	Synaptotagmin-1 R. Henderson, A.                Periplasmic	 Chaperone	 Skp	 and	 Hybrid	 Structure	 of	
Daily and T.K. S. Kumar. Univ. of Arkansas.                          the Periplasmic Domain of BamA M. Sousa, A. Pardi,
                                                                     T. Walton, P. Gatzeva-Topalova and C. Sandoval. Univ. of
                                                                     Colorado at Boulder.

                                     WEDnESDAy,	AUGUST	4
B120 559           The Folding and Energy Landscapes of
Spectrin R15, R16 and R17 B. Wensley, L.G. Kwa, S. Batey,             Protein Structure/Function
Z.M. Chan, N. Tumelty, F. Bone, A. Steward, A. Borgia and
J. Clarke. The Scripps Res. Inst. and Univ. of Cambridge.
B121 560           IkBa Structure and Dynamics Explored               B130 569	          The	 Varied	 Roles	 of	 Highly	 Conserved	
by FRET J. Lamboy, H. Kim, T. Ha and E. Komives. UCSD                 Residues	 near	 or	 within	 Beta-Turns	 in	 Glyoxysomal	
and Univ. of Illinois at Urbana-Champaign.                            Malate Dehydrogenase S. Hedrick and E. Bell. Univ. of
B122 561	          ATP	Functions	as	a	Chemical	Chaperone	             Richmond.
for	 the	 Folding	 of	 Glyceraldehyde-3-Phosphate	                    B131 570	          Structure	 of	 the	 SCAn	 Domain	 from	
Dehydrogenase C. Park and P-F. Liu. Purdue Univ.                      the	Zinc	Finger	Transcription	Factor	Zfp206 Y. Liang, G.
B123 562	          Dodging	 the	 Crisis	 in	 Protein	 Folding	        Pugalenthi, P. Palasingam and P.R. Kolatkar. Genome Inst. of
with Knots J. Sulkowska, J. Noel, P. Sulkowski, P. Vrinau             Singapore.
and J. Onuchic. UCSD, Caltech and Johannes Gutenberg                  B132 571           Insight Into the Mechanisms of Notch1
Univ. Mainz, Germany.                                                 Activation and Inhibition K. Tiyanont, T. Wales, J. Engen
B124 563	          Folding,	 Conformation,	 and	 Stability	           and S. Blacklow. Brigham and Women’s Hosp., Harvard
of	yeba-Like	Protein	Structures:	Proteins	with	Knots M.               Med. Sch. and Northeastern Univ.
Roy, D. Capraro, C. Cole, B. Wong and P. Jennings. UCSD.              B133 572           ATP Modulates the Oligomerization of
                                                                      Chloroplast	2-Cys	Peroxiredoxin M. Aran, D. Ferrero and
Protein Formulation and                                               R. Wolosiuk. Inst. Leloir Fndn., Buenos Aires.
                                                                      B134 573           Protein Structure Initiative Material
Process Development                                                   Repository:	A	Resource	of	Structural	Genomics	Plasmids	
                                                                      for	 the	 Biological	 Community C. Cormier and J. LaBaer.
B125 564           Formulation Strategies for Stabilization
                                                                      Arizona State Univ.
of a Monoclonal Antibody Solution: Towards the
                                                                      B135 574           Evolution of New Enzymatic Function
Development	 of	 Dilute	 and	 a	 Highly	 Concentrated	
                                                                      by	 Structural	 Modulation	 of	 Cysteine	 Reactivity	 in	
Immunoglobulin	 G	 Formulations	 for	 Subcutaneous	
                                                                      Isocyanide Hydratase P. Madzelan, M. Lakshminarasimhan,
Administration A. Bhambhani, J. Kissmann, R. Kashi, S.
                                                                      R. Nan, N. Milkovic and M. Wilson. Univ. of Nebraska-
Joshi and C.R. Middaugh. Merck & Co. Inc., West Point, PA
and Summit, NJ and Univ. of Kansas.
                                                                      B136 575           Identifying Melanogenesis Inhibitors
B126 565           Application of Extrinsic Fluorescence
                                                                      by Targeting the Human Tyrosinase from Cinnamomum
Spectroscopy for the High Throughput Screening of
                                                                      subavenium with in Vitro and in Vivo Screening Systems
Stabilizers of Aluminum-Adjuvanted Protein Vaccines S.
                                                                      H-M. Wang. Kaohsiung Med. Univ., Taiwan.
Ausar, J. Chan, W. Hoque, O. James, K. Jayasundara and K.
                                                                      B137 576           Non-essential A69E Mutation in the
Harper. Sanofi Pasteur, Toronto.
                                                                      Active Site of Aspergillus niger PhyA Phytase Leads
B127 566	          Protein	Effective	Charge	in	the	Presence	
                                                                      to	 Myriads	 of	 Biochemical	 Changes A. Ullah, K.
of	 neutral	 Salts:	 Anions	 Influence	 Protein	 Aggregation	
                                                                      Sethumadhavan, S. Boone and E. Mullaney. USDA, New
through Direct Interactions with the Protein Surface
R. Fesinmeyer, S. Hogan, V. Razinkov, M-K. Reynolds, A.
                                                                      B138 577           Increased Binding to Triglyceride-
Saluja, Y. Gokarn, G. Becker, L. Narhi and D. Brems. Amgen
                                                                      Rich	 Lipoproteins	 and	 Loosely	 Folded	 Conformation	
Inc., Seattle.
                                                                      of Apolipoprotein E4 and A-I Forms That Induce
B128 567           Automated        Measurement         and
                                                                      Hypertriglyceridemia I. Gorshkova, V.I. Zannis and D.
Comparison	of	Protein	Conformation D. Gregson and L.
                                                                      Atkinson. Boston Univ. Sch. of Med.
Cole. Applied Photophysics Ltd., Leatherhead, U.K.
                                                                      B139 578           The Study on Membrane-Bound Alpha-
B129 568           Mechanisms of Protein Aggregation
                                                                      Synuclein	and	Its	Parkinson’s	Disease	Linked	Mutants	by	
by Antimicrobial Preservatives K. Mallela, S. Singh,
                                                                      Pulse Dipolar EPR Spectroscopy E. Georgieva, T. Ramlall,
J. Cabello-Villegas and R. Hutchings. Univ. of Colorado
                                                                      P. Borbat, D. Eliezer and J.H. Freed. Cornell Univ. and Weill
Denver, Aurora.
                                                                      Cornell Med. Col.
                                                                      B140 579	          Structural	 Genomics	 of	 Eukaryotic	
                                                                      Chaperone	 Proteins J. Osipiuk, M. Gu, R. Mulligan, M.
                                                                      Zhou, N. Maltseva, E. Duggan, H. Li, C. Hatzos, M. Mihelic,
                                                                      M. Bargassa, P. van Oosten-Hawle, C. Voisine, C. Sahi, E.
                                                                      Craig, R.I. Morimoto and A. Joachimiak. Argonne Natl. Lab.,
                                                                      Northwestern Univ. and Univ. of Wisconsin-Madison.

                                     WEDnESDAy,	AUGUST	4
B141 580           Structural Basis of Bro1 Domain                   B154 593	        Structural	Transitions	in	Class	II	HMG-
Interaction	 with	 the	 ESCRT	 Machinery	 and	 Its	 Role	 in	        CoA	 Reductase	 Function J. Watson, H. Dembinski, A.
HIV Budding R. Mu, V. Dussupt, P. Sette, G. Snyder, F.               Commeree, J. Driver and H. Hanks. Gonzaga Univ., WA.
Bouamr and T. Xiao. NIAID, NIH.                                      B155 594         Structure      of    Ferredoxin-NAD(P)+
B142 581	          Preparation	 of	 Human	TrkB	 Domain	 5	           Oxidoreductase	 from	 the	 Green	 Sulfur	 Bacterium	
and	 Preliminary	 Characterization	 of	 Its	 Structure	 and	         Chlorobaculum tepidum: Implications for Binding
Flexibility Using Heteronuclear NMR K. Crowhurst and                 Ferredoxin and NADP+ G. Kurisu, N. Muraki, T. Sakurai
W.H. Kim. California State Univ., Northridge.                        and D. Seo. Osaka Univ., Univ. of Tokyo and Kanazawa
B143 582           Structural Study of Helicobacter pylori           Univ.
HP1454 Secreted Protein S. Quarantini, L. Cendron and G.             B156 595         Structural and Biochemical Study of
Zanotti. Univ. of Padua and Venetian Inst. of Molec. Med.            Herpes Virus Proteins F. Nasertorabi, S-L. Dahlroth, R.
B144 583           Helicobacter pylori Acidic Stress                 Venkatachalam, J. Karlsson, K. Yeung, D. Gurmu and P.
Response Factor HP1286 Is a YceI Homolog with New                    Nordlund. Nanyang Technol. Univ., Singapore and Karolinska
Binding	Specificity L. Sisinni, L. Cendron and G. Zanotti.           Inst.
Univ. of Padua, Italy and Venetian Inst. of Molec. Med.              B157 596	        The	Crystal	Structure	of	the	Exocellulase	
B145 584	          Determinants	of	Cdk	Complex	Assembly	             Cel6B	(E3)	from	Thermobifida fusca H. Hansson, M. Wu,
by RXL Motif Peptides S. Otieno and R. Kriwacki. St. Jude            S. Karkehabadi, B. Keleman, E. Larenas, C. Mitchinson and
Children’s Res. Hosp. and Univ. of Tennessee Hlth. Sci. Ctr.,        M. Sandgren. Swedish Univ. of Agr. Sci. and Danisco US
Memphis.                                                             Inc., Genencor Div., Palo Alto.
B146 585	          Structural	 Characterization	 of	 the	            B158 597         Aqueous Salt Solution of Organic
Psuedouridine N1-Methyltransferase, Nep1, Bound to                   Solvents as a Media for Enzymatic Reactions B. Minofar,
RNA — Insight into Its Role in Ribosome Biogenesis                   M. Khabiri, J. Damborsky and R. Ettrich. Inst. of Syst. Biol.
N. LaRonde-LeBlanc, S. Thomas and C. Keller. Univ. of                and Ecol., Acad. of Sci. of Czech Republic, Univ. of South
Maryland College Park.                                               Bohemia and Masaryk Univ., Czech Republic.
B147 586           A Target Protein Recognition System of            B159 598	        Structural	 Characterization	 of	 new	
Human	 Cbl	 Ubiquitn	 E3	 Ligase	 Family K. Takeshita, T.            Family of Tetrameric Flavoproteins WrbA M. Kuty, J.
Tezuka, E. Yamashita, M. Suzuki and A. Nakagawa. Osaka               Wolfova, J. Brynda, J. Mesters, R. Ettrich, J. Carey and I.
Univ. and Univ. of Tokyo.                                            Kuta Smatanova. Inst. of Syst. Biol. and Ecol., Acad. of Sci.
B148 587	          Cloning,	 Over-Expression,	 Purification,	        of Czech Republic, Univ. of South Bohemia, Inst. of Molec.
Crystallization,	 and	 Biochemical	 Study	 of	 Human	                Genet., Prague, Univ. of Lübeck, Germany and Princeton
NH2-Terminal	 Truncated	 TCTP	 with	 IgE-Dependent	                  Univ.
Histamine-Releasing Activity M-S. Kim and D.H. Shin.                 B160 599	        The	 Crystal	 Structure	 of	 an	 Improved	
Ewha Womans Univ., South Korea.                                      Thermal	Stability	Variant	of	Cellobiohyrolase	2	(Cel6A)	
B149 588	          Crystal	 Structure	 of	 the	 PTEn-Like	           from Hypocrea jecorina S. Karkehabadi, M. Sandgren, F.
Region	of	Ci-VSP M. Matsuda, S. Sakata, K. Takeshita, M.             Goedegebuur, E. Larens and C. Mitchinson. Swedish Univ.
Suzuki, E. Yamashita, Y. Okamura and A. Nakagawa. Inst. of           of Agr. Sci., Danisco, Genencor BV, Leiden and Danisco,
Protein Res. and Grad. Sch. of Med., Osaka Univ.                     Palo Alto.
B150 589           Effect of Familial Hypobetalipopro-               B161 600	        Crystallographic	 Studies	 of	 an	
teinemia Point Mutations on the Structure of the                     Aminopeptidase, ERAP1, That Is Involved in Antigen
N-Terminal Region of Apolipoprotein B L.E. Packer and                Processing T. Nguyen and L. Stern. Univ. of Massachusetts
C.J. McKnight. Boston Univ. Sch. of Med.                             Med. Sch.
B151 590           Stereochemistry       Determinants     of         B162 601	        A	 new	 Class	 of	 2Fe-2S	 Proteins	 from	
3-Hydroxy-3-Methylglutaryl	Coenzyme	A	in	Enterococcus                the Plant Arabidopsis thaliana M. Paddock, A. Conlan,
faecalis	HMG-CoA	Synthase Y.C. Chang, A. Sutherlin, C.               O. Yogev, Y. Harir, E. Abresch, P. Jennings, R. Mittler and
Steussy, V. Rodwell and C. Stauffacher. Purdue Univ. and             R. Nechushtai. UCSD, The Hebrew Univ. of Jerusalem and
Abilene Christian Univ.                                              Univ. of Nevada, Reno.
B152 591	          Conformationally-Gated	          Electron	        B163 602	        Solution	 Conformation	 of	 the	 HIV-1	
Transfer	Reactions	in	yeast	Iso-1-Cytochrome	c: Effects              Integrase	Catalytic	Core	Domain:	A	Magnesium-Induced	
of	Mutagenesis,	Chemical	Reductants,	and	pH M. Cherney,              Structural Transition N. Fitzkee, J. Masse, Y. Shen, D.
C. Junior and B. Bowler. Univ. of Montana.                           Davies and A. Bax. NIDDK, NIH.
B153 592           Structural Investigation of the Iron-             B164 603	        Characterization	of	Tyrosine	and	Amino-
Regulated Surface Determinant Protein IsdB B. Barry, B.              Tyrosine Radicals in Azurin V. Mosser, M. Machczynski
Tripet, B. Lei and V. Copie. Montana State Univ.                     and M. McGuirl. Univ. of Montana and Yeshiva Univ.
                                                                     B165 604	        novel	 Stabilizer/Blocker	 Formulations	

                                  WEDnESDAy,	AUGUST	4
Providing	 Rare	 Dual	 Abilities	 to	 Block	 Immunoassay	         B168 607          Interfacial Activation of Human
Diagnostic Surfaces and Prevent Protein Denaturation for          Secreted Phospholipase A2 Enzymes J. Urbauer, K. Nemec,
Effective Long-Term Dried Stability T. Jentz. SurModics,          S. Ray and S. Tatulian. Univ. of Georgia and Univ. of Central
Eden Prairie, MN.                                                 Florida.
B166 605         Direct Observation of the Multiple               B169 608          Structural         and         Functional
Structures Involved in Protein Kinase Function M.                 Characterization	 of	 Glycine	 Rich	 RnA	 Binding	 Protein	
Jamros, L. Oliveira, P. Whitford, J. Onuchic, J. Adams, D.        Involved in Senescence of Barley K. Mason, B. Tripet, A.
Blumenthal and P. Jennings. UCSD, Los Alamos Natl. Lab.           Fischer and V. Copie. Montana State Univ.
and Univ. of Utah.                                                B170 609          New Roles for the Anti-Sigma Factor
B167 606	        Crystal	Structure	of	Miner1	Reveals	the	         AsiA in RNA Polymerase Regulation A. Dery, R. Bieber
Protein	Causative	in	Wolfram	Syndrome	2	Is	Homodimeric	           Urbauer, H. Tao and J. Urbauer. Univ. of Georgia.
with	 Rare	 Coordination	 of	 the	 Redox	 Active	 2Fe-2S	
Clusters A. Conlan, M. Paddock, H. Axelrod, A. Cohen, E.
Abresch, J. Zuris, R. Nechushtai and P. Jennings. UCSD,
Stanford Synchrotron Radiation Lab. and Hebrew Univ. of

     24th SympoSium of the protein Society
               AuguSt 1–5, 2010
           manchester grand hyatt San Diego
            ground floor / Douglas pavilion
                San Diego, california

                                      Douglas Foyer

                          Ground Floor/Douglas Pavilion at the Manchester Grand Hyatt San Diego
                              Exhibit Days and Hours: Monday, August 2, 12:00 PM – 6:45 PM
                                                      Tuesday, August 3, 10:00 AM – 6:45 PM
                                                      Wednesday, August 4, 10:00 AM – 3:30 PM

   Anton PAAR USA                                            303         Bio-Logic USA                                             318
   10215 Timber Ridge Drive                                              PO Box 30009
   Ashland, VA 23005                                                     Knoxville, TN 37930
   Phone: 800 722-7556                                                   Phone: 865 769-3800
   Fax: 804 550-1057                                                     Fax: 865 769-3801
   Email:                                  Email:
   Web:                                               Web:
   Anton Paar produces high-end instruments for materials                Bio-Logic USA is a major supplier of stopped-flow, quench-
   science for understanding and optimizing material properties.         flow, and freeze quench instruments used for rapid kinetic
   The SAXSess mc² is a new measuring system for nanostructure           studies in chemistry and molecular biology. The systems are
   analysis using small-angle X-ray scattering. It is a universal        designed for the highest possible performance, flexibility,
   tool for investigating nanostructures in different materials,         and versatility. Sophisticated hardware and software
   including proteins.                                                   allow experiments to be designed from the keyboard, and
                                                                         the instruments to be configured for new applications in
   Applied Photophysics                                      319         minutes.
   203-205 Kingston Road
   Leatherhead, Surrey KT22 7PB                                          Dionex Corporation                                        317
   United Kingdom                                                        1228 Titan Way, PO Box 3603
   Phone: 800 543-4130                                                   Sunnyvale, CA 94088-3603
   Fax: 44 1372 386477                                                   Phone: 408 737-0700
   Email:                                         Fax: 408 730-9403
   Web:                                             Email:
   Applied Photophysics Ltd is a world-leading manufacturer of
   precision spectrometers used for the study of the structural,         Dionex Corporation is a leading supplier of liquid
   thermodynamic and kinetic properties of a wide range                  chromatography supplies for biomolecules separation. The
   of samples. Our product range includes: - Chirascan and               Dionex ProPac and ProSwift ion-exchange, HIC, IMAC
   Chirascan-plus circular dichroism spectrometers - SX20                and RP columns are the industry standards for proteins
   stopped-flow spectrometer - LKS.60 laser flash photolysis             characterizations. The Dionex UltiMate® 3000 Titanium
   spectrometer.                                                         is a fully biocompatible HPLC with unique dual gradient
                                                                         pump design and fraction collection re-injection option in the
H Aviv Biomedical, Inc. — Silver Corporate Member            213         autosampler that allows full automation of multiple separation
   750 Vassar Avenue, Suite 2                                            steps that is typical in protein separation.
   Lakewood, NJ 08701-6929
   Phone: 732 370-1300
   Fax: 732 370-1303
   Aviv Biomedical Inc. manufactures scientific and clinical
   instruments.Products include a fluorescence accessory (AU-
   FDS) for the Beckman Analytical Ultracentrifuge, model
   XLA/XLI. Sales, service, and support of Aviv spectrometers,
   Aviv spectrophotometers and Aviv fluorometers.

DNA2.0                                                   203         Fluid Imaging Technologies, Inc.                           210
1430 O’Brien Drive, Suite E                                          65 Forest Falls Drive
Menlo Park, CA 94025                                                 Yarmouth, ME 04096
Phone: 650 853 8347                                                  Phone: 207-846-6100
Fax: 650 618 2697                                                    Email:
Email:                                                Web:
                                                                     FIT manufacturers FlowCAM®, a continuous-imaging particle
Gene synthesis company DNA2.0 is the fastest provider                analyzer that rapidly characterizes particles in formulations.
of synthetic genes and the leader in codon optimization              FlowCAM is highly sensitive to translucent particles and low
research. DNA2.0 explores novel applications for synthetic           concentrations. Since it images each particle characterized, it
genes and is exploiting the synergy between highly efficient         enables users to classify and quantify silicone oil droplets or
gene synthesis processes and new protein engineering and             protein aggregates. Configurations available for visible and
optimization technologies. For more information, please visit        sub-visible particle characterization.
                                                                     ForteBio                                                   202
Elsevier                                                 204         1360 Willow Road, Suite 201
360 Park Avenue South                                                Menlo Park, CA 94025
New York, NY 10010                                                   Phone: 650 289-6800
Phone: 212 989-5800                                                  Fax: 650 322-1330
Fax: 212 633-3112                                                    Email:
Web:                                                Web:
As the world’s leading publisher of science and health               ForteBio markets a novel, label-free, 96 or 384-well format
information, Elsevier serves more than 30 million scientists,        detection platform that includes instrument hardware,
students, and health and information professionals globally.         biosensors and data analysis software to measure affinity,
By delivering world-class information and innovative tools to        kinetics, and concentration in crude or purified samples. This
researchers, students, educators and practitioners worldwide,        real-time dip and read method allows greater throughput and
we help them increase their productivity and effectiveness.          cost-effectiveness in many applications compared to existing
                                                                     methods such as SPR, ELISA and HPLC.
EMD Chemicals Inc.                                       218
10394 Pacific Center Ct.                                             GE Healthcare                                              307
San Diego, CA 92121                                                  800 Centennial Avenue, PO Box 1327
Phone: 858 450-9600                                                  Piscataway, NJ 08855
Fax: 856 453-3552                                                    Phone: 800 526-3593
Email:                               Fax: 877 295-8102
Web:                                            Email:
FASEB                                                    201         GE Healthcare is ready to help meet your protein challenges.
9650 Rockville Pike                                                  The Life Sciences division offers a range of products and
Bethesda, MD 20814                                                   services from protein sample preparation and purification
Phone: 301 634-7930                                                  to protein characterization. Our products offer a complete
Fax: 301 634-7353                                                    solution, now including the Biacore and MicroCal systems.
Web:                                                   HORIBA Scientific                                          304
FASEB MARC Program provides a variety of activities to               3880 Park Avenue Edison
support the training of minority students, postdoctorates,           Edison, NJ 08820
faculty and scientists in the biomedical and behavioral              Phone: 732.494.8660
sciences. We offer travel awards for scientific meetings,            Fax: 732.549.5125
research conferences, and student summer research                    Email:
opportunities programs. We also sponsor Career Development           Web:
Programs including grantsmanship training seminars.                  Horiba Scientific is a worldwide leader in spectroscopy and
                                                                     molecular analysis. Surface plasmon resonance imaging
                                                                     technology from Horiba Genoptics offers multiplex
                                                                     characterization of protein, antibody, DNA, RNA, and small
                                                                     molecule binding kinetics with up to 400 interactions in one

  ICx Nomadics                                              107         Malvern Instruments                                         310
  800 Research Parkway, Suite 100                                       117 Flanders Road
  Oklahoma City, OK 73104                                               Westborough, MA 01581
  Phone: 405 239-8600                                                   Phone: 508 768-6400
  Fax: 405 235-8608                                                     Fax: 508 768-6403
  Email:                                               Email:
  Web:                                          Web:
  ICx Nomadics offers a complete line of Surface-Plasmon-               Malvern’s material characterization tools deliver
  Resonance instruments for Label-Free analysis of                      measurements reflecting the complexities of macromolecules,
  biomolecules.Our specialty is high-quality affinity and               nanomaterials, particulates and disperse systems. Molecular
  kinetic analysis of small molecules using medium-to-high              size, particle shape, zeta potential, molecular weight, chemical
  throughput at affordable prices. ICx Nomadics is located in           composition and rheological properties measurements are
  Oklahoma City, OK.                                                    now joined by advanced chromatography solutions (GPC/
                                                                        SEC), extending Malvern’s technologies for protein molecular
  INTAVIS Inc.                                              306         weight, size and aggregation measurements.
  945 W George St, Suite 201
  Chicago, IL 60657                                                     Molecular Dimensions                                        300
  Phone: 773 572-5799                                                   PO Box 1001
  Fax: 773 305-1845                                                     Apopka, FL 32704
  Email:                                            Phone: 813-943-5232
  Web:                                                  Fax: 866-283-8330
  INTAVIS Bioanalytical Instruments Is an internationally active        Web:
  company offering instruments for biochemical applications.
  We concentrate on customer-oriented automation of complex             Molecular Dimensions is dedicated to supplying products for
  procedures in multiple parallel peptide synthesis, proteomics         biomolecular structure research. We specialize in supplies
  and functional genomics. Our new peptide synthesis service            for protein crystal growth, such as screening kits, plates,
  offers custom Peptide Sets and both standard and custom               coverslides and robotics. Our aim is to provide the materials
  CelluSpot peptide microarrays.                                        and products needed for biomolecular structure quickly and
H JASCO — Silver Corporate Member                           302
  28600 Mary’s Court                                                    Olink Bioscience                                            206
  Easton, MD 21601                                                      6181 Cornerstone Court East, Suite 103
  Phone: 410 822-1220                                                   San Diego, CA 92121
  Fax: 410 822-7526                                                     Phone: 800 900-0065
  Email:                                             Email:
  Web:                                                 Web:
  JASCO is the world leader in Circular Dichroism Spectroscopy.         Olink Biosciences is focused towards the development
  With over 50 years of advanced manufacturing and design               of cutting-edge technologies for biomolecular analysis in
  capabilities JASCO’s new Model J-815 is a real workhorse              research and diagnostics. The proprietary Duolink® product
  capable of measuring CD, Absorbance and Fluorescence as               line enables visualization and accurate quantification of
  well as stopped-flow in all three modes. Stop by Booth #302           individual proteins, their interactions and modifications, in
  for a quick demonstration.                                            unmodified cells and tissues. For more information visit:

Photon Technology International, Inc.                      101         Reichert Technologies - Life Sciences                      219
P. O. Box 272                                                          3362 Walden Avenue
Birmingham, NJ 08011-0272                                              Depew, NY 14043
Phone: 609 894-4420                                                    Phone: 716 686-4513
Fax: 609 894-1579                                                      Fax: 716 686-4554
Email:                                            Email:
Web:                                                    Web:
PTI’s QuantaMaster is a high performance spectrofluorometer            Reichert SPR instruments are ideal for investigating label-free
with an open architecture design.Its signal-to-noise ratio of          molecular interactions in real-time. Our modular, component-
10000:1 makes it the most sensitive steady state fluorometer           based systems offer the most flexible platform on the market.
on the market! It accommodates a fluorescence lifetime option,         As a scientific research partner and teaching tool, Reichert
programmable sample temperature controller, polarizers, etc.           SPR systems offer outstanding value with low maintenance
It’s ideal for studying protein folding/unfolding and protein          at an affordable price.
                                                                       Sarstedt, Inc.                                             111
The Protein Society                                        103         PO Box 468
9650 Rockville Pike                                                    Newton, NC 28658
Bethesda, MD 20814                                                     Phone: 828 465-4000
Phone: 301 634-7277                                                    Fax: 828 465-0718
Fax: 301 634-7271                                                      Email:
Email:                                   Web:
                                                                       The Sarstedt Group develops, manufactures, and markets
The Protein Society is the leading international society               equipment and consumables for medicine and research.
devoted to furthering research and development in protein              The company’s range of laboratory products is the result of
science. Our mission is to advance state-of-the-art science            in-house design and stringent quality control procedures.
through international forums that facilitate communication,            Products for the research laboratory include cell culture
cooperation and collaboration among scientists involved                labware; consumables for PCR, molecular biology, and
in the study of proteins. The Society publishes Protein                cryopreservation; and benchtop instruments.
Science, the premier journal in the field. Members have an
opportunity to actively participate in the emerging fields of          Sierra Sensors GmbH                                        119
protein science, such as proteomics, bioinformatics, structural        7 Austin Avenue, Suite 5
biology and computational biology as they pertain to proteins          Greenville, RI 02828
at the molecular and cellular level. Our members represent a           Phone: 401 404-5549
wide range of professionals working in academia, industry,             Email:
government and non-profit institutions seeking to explore              Web:
new frontiers in biology, medicine and technology. Stop by
The Protein Society booth to discuss your membership, ideas            Sierra Sensors is combining state-of-the-art label-free
or suggestions with the Executive Officer, Cindy Yablonski,            detection with cutting edge microfluidic sample delivery,                                         sensor design, and automation, to supply researchers at all
                                                                       levels with high performance analytical biosensors that are
Randox Laboratories Ltd                                    311         practical to own and operate.
55 Diamond Road
Crumlin, Co. Antrim BT29 4QY                                           Springer                                                   305
United Kingdom                                                         233 Spring Street
Phone: +44 (0)28 944 22413                                             New York, NY 10013
Email:                                           Phone: 1-800-777-4643
Web:                                                    Fax: 212 463-0742
Randox Laboratories is an international diagnostic solutions           Web:
company, revolutionising healthcare worldwide. Our product
portfolio is extensive, including clinical chemistry reagents          Springer is a major publisher of books and journals in Life
and analysers, life sciences, quality control material in              Sciences. Please stop by our booth to order books at a special
conjunction with EQA and IQC schemes, and revolutionary                conference discount and take a closer look at sample issues
Biochip Array Technology for simultaneous detection of                 of journals. Staff will be available to answer any questions
multiple analytes from a single sample for rapid and reliable          about publishing with Springer.

  St. Jude Children’s Research Hospital                       301         Wiley Blackwell                                         211
  262 Danny Thomas Place, MS 276                                          111 River Street
  Memphis, TN 38105-2794                                                  Hoboken, NJ 07030
  Phone: 901 595-2750                                                     Phone: 201-748-8893
  Fax: 901 595-5376                                                       Fax: 201-748-6617
  Email:                                           Web:
                                                                          Wiley-Blackwell is the international scientific, technical,
  St Jude Children’s Research Hospital is a non-profit biomedical         medical and scholarly publishing business of John Wiley
  research institution in Memphis, TN, where 190 basic science            & Sons, with strengths in every major academic and
  and clinical researchers investigate the molecular basis of             professional field and partnerships with many of the world’s
  both normal cellular and diseased processes. Visit our booth            leading societies. For more information, please visit www.
  to discuss and apply for Postdoctoral Fellowship positions     or
  for PhD, MD, PharmD, and DVM students.
                                                                     H Wyatt Technology — Silver Corporate Member                 200
H Thermo Scientific — Gold Corporate Member                   207         6300 Hollister Avenue
  P.O. Box 117                                                            Santa Barbara, CA 93117
  Rockford, IL 61101                                                      Phone: 805 681-9009
  Phone: 815 968-0747                                                     Fax: 805 681-0123
  Fax: 815 968-7316                                                       Email:
  Email:                                              Web:
                                                                          DAWN multi-angle (MALS) and DynaPro dynamic light
  Thermo Scientific Pierce Protein Research Products include              scattering (DLS) instruments for determining absolute
  kits and reagents for sample preparation; protein detection and         masses and sizes of proteins and conjugates in solution.
  quantitation, purification, interaction, and function studies;          These systems are used in stand-alone mode, or as on-line
  and custom antibody and peptide services. Pierce Human in               detectors connected to HPLC separation technology to
  vitro Protein Expression Kits, protein assays, electrophoresis          study oligomerization (assembly states), protein-protein
  and Western blotting products are essentials tools for efficient        interactions, and protein crystallography conditions. In
  life science research.                                                  addition, we introduce the world-class Möbiuz mobility
                                                                          instrument whose capabilities extend far beyond conventional
  Thermo Scientific, NanoDrop products                        205         zeta-potential measurements.
  3411 Silverside Road, Bancroft Building, Suite 100
  Wilmington, DE 19810
  Phone: 302 479-7707
  Fax: 302 792-7155
  Thermo Scientific NanoDrop products are specifically
  designed for 1µl samples, employing a patented retention
  system that eliminates the need for dilutions of sample.
  Using this novel system, the Thermo Scientific NanoDrop™
  2000 and 2000c spectrophotometers and the NanoDrop 8000
  spectrophotometer enable full UV-Vis absorbance analysis.

                  Participation in the Exhibits Program does not constitute an endorsement by
                         The Protein Society of the claims, products, or services offered.

                                        EXHIBITOR WORKSHOPS

                                              Monday, August 2
Structural Basis of Shh Regulation
by Neutralizing Antibody 5E1 and
Hedgehog-Interacting Protein: Role of
the Shh Pseudo-Active Site in Signaling
1360 Willow Road, Suite 201
Menlo Park, CA 94025
Phone: 650 322-1360
Fax: 650 322-1370

12:15 PM – 1:15 PM – Manchester E

Presenter: Henry R. Maun, PhD, Genentech Inc.

Sonic hedgehog (Shh) signaling is important for proper
cellular differentiation during embryogenesis and mostly
quiescent in adults, but dysregulation of this pathway
has been implicated in cancer, where inhibition shows
therapeutic benefit. The Shh-neutralizing antibody 5E1 and
receptor Hedgehog-interacting protein (Hhip) are potent
inhibitors of Hh signaling. We solved crystal structures of
chimeric 5E1(ch5E1) Fab and Hhip, each complexed with
Shh, which revealed the pseudo-active site groove on Shh
as common binding site. The maximal binding affinities
of Hhip and 5E1 for Shh depend on Zn2+ and Ca2+ being
bound to the pseudo-active site and mutations in this site are
linked to the genetic disease BDA1.

                                            Tuesday, August 3
Protein Characterization with Light                                Duolink – Discover More About Protein-
Scattering                                                         Protein Interactions
Wyatt Technology Corporation                                       Olink Bioscience
6300 Hollister Avenue                                              6181 Cornerstone Court East
Santa Barbara, CA 93117                                            San Diego, CA 92121
Phone: 805-681-9009                                                Phone: 800 900-0065
Fax: 805-681-0123                                                  Email:
Email:                                              Web:
                                                                   12:00 PM – 1:00 PM – Manchester E
12:00 PM – 1:00 PM – Manchester F
                                                                   Presenter: Bonnie Tran
Presenter: Sigrid Kuebler
                                                                   Olink Bioscience offers a new and enabling research tool,
The seminar will provide a brief introduction to light             Duolink(®), based on the in situ PLA(®) technology
scattering and an understanding of how our DAWN family of          for detection of proteins in fixed cells or tissue. Duolink
Multi-Angle Light Scattering (MALS) and DynaPro family             enables direct visualization of protein-protein interactions,
of Dynamic Light Scattering (DLS) detectors can determine          post-translational modifications and single proteins with
absolute molar masses, sizes, and conformations of proteins        unparalleled specificity and sensitivity using a standard
and protein conjugates in solution. Also provided will be          fluorescence or bright field microscope. Target proteins are
information on realizing fast and reliable measurements of         detected with single molecular resolution, giving information
macromolecular electrophoretic mobilities with our Möbius          about the exact location of the targets within the cells or tissue
instrument, specifically designed to address the unique            sample and allowing digital quantification. Furthermore, the
measurement of protein mobilities.                                 proteins can be studied at physiological expression levels,
                                                                   eliminating the need for over-expression or modifications
                                                                   of the proteins. These unique and enabling features make
                                                                   Duolink a powerful tool to study signal transduction
                                                                   pathways and elucidate disease mechanisms, leading to the
                                                                   development of novel diagnostics and therapeutics. In this
                                                                   seminar the principle of the innovative technology behind
                                                                   Duolink will be presented along with some captivating
                                                                   customer application examples. The listeners will be exposed
                                                                   to a new way of studying.

                                           Wednesday, August 4
SensiQ Pioneer — The New Kid on the
Label-Free Interaction Analysis Block
ICx Nomadics
800 Research Parkway, Suite 100
Oklahoma City, OK 73104
Phone: 405-239-8600
Fax: 405-235-8608

12:30 PM – 1:30 PM – Manchester D

Presenters: David Myszka, PhD, Biosensor Tools
            John Quinn, PhD, ICx Nomadics

David Myszka, PhD of Biosensor Tools and John Quinn,
PhD of ICx Nomadics will present SensiQ Pioneer, a Surface
Plasmon Resonance - based biomolecular interaction analysis
platform produced by ICx Nomadics. Drs. Myszka and Quinn
will present data taken using Fast-StepTM Gradient Injection,
a unique method for enabling high throughput screening.

        Exhibitor Workshops are open to all registered scientific participants. The sponsoring
     company has the option, at its sole discretion, to refuse admittance to nonscientific attendees.



Abstract Number

1 through 58                               Invited Speaker Oral Presentations

100 through 269                            Poster Session 1 – Monday, August 2, 1:15 PM – 2:45 PM

270 through 439                            Poster Session 2 – Tuesday, August 3, 1:15 PM – 2:45 PM

440 through 609                            Poster Session 3 – Wednesday, August 4, 1:45 PM – 3:15 PM

Invited Speakers (1 – 58)                                                be difficult. Directed evolution is a quick and easy method
                                                                         of finding mutations that result in increased solubility.
                                                                         The difficult step in this process is selection. In this talk a
                                                                         particularly insensitive method is described for monitoring
Electrostatic Effects in Proteins: Charges and                           protein solubility. It involves fusing mutant proteins to
Consequences                                                             dihydrofolate reductase and selecting for soluble variants
J. Martin Scholtz                                                        by plating transformants on plates containing Trimethoprim.
Texas A&M University, Molecular & Cellular Medicine,                     A number of test cases will be described that resulted in
College Station, TX, 77843-1112                                          dramatically increased levels of protein solubility.
Many fundamental properties of proteins depend strongly
on the pH and other characteristics of the solution including            3
stability, solubility and even the conformation of the                   DNA repair machines, disease causing mutations, and
polypeptide chain. Many of these properties are affected by              insights for cancer interventions
the interactions between charges of the ionizable groups of              John Tainer
the polypeptide chain, both inter- and intramolecular effects            Molecular Biology, The Scripps Research Institute, 10550
are important. Here we will describe our use of a series of              North Torrey Pines Road, MB4, La Jolla, CA, 93037
ribonuclease proteins that differ principally in the net charge
on their surface to help understand the role that electrostatic          DNA repair machines prevent DNA double-stand breaks
interactions play in modulating a number of important                    (DSBs) from becoming chromosome breaks by tethering
properties of proteins and polypeptides. Our series of proteins          the DNA ends and by orchestrating signaling, chromatin,
span a range of pI from less than 3 to over 10 and thus we have          and repair responses. The Mre11-Rad50-Nbs1 (MRN) is a
a novel system that can be used to address several different             keystone complex that connects DNA repair machinery,
properties of proteins that depend on net charge, pH and the             double-strand break signaling, and the chromatin template.
interplay of electrostatic interactions.                                 To organize the MRN complex, the Mre11 exonuclease
                                                                         directly binds Nbs1, DNA and Rad50. Mre11 dimers bind
                                                                         and distinguish two-ended DSBs and collapsed replication
                                                                         forks (Williams et al., 2008). Rad50 employs its ATP-binding
Improving protein solubility: an evolutionary approach                   cassette (ABC) ATPase, Zn-hook and coiled-coils to bridge
using E.coli DHFR as a reporter                                          DSBs and facilitate DNA end processing by Mre11. The
David Ollis                                                              Nijmegen breakage syndrome 1 (Nbs1) subunit coordinates
Research School of Chemistry, Australian National                        DSB repair and checkpoint signaling through interactions with
Univversity, Australian National University, Research School             ATM, MDC1 and Sae2/Ctp1/CTIP via its fused, extended,
of Chemistry, Building 35, Canberra, ACT 0200, Canberra,                 FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal
0200, Australia                                                          Mre11- and ATM-binding motifs. These interactions promote
Low protein solubility is frequently a problem in the                    central architectural, structural, enzymatic, sensing, and
characterisation and utilisation of proteins. There are a variety        signaling functions in DSB repair (Williams et al., 2009).
of in vitro physical and chemical techniques that can be used            Structural and biological evidence suggests MRN has three
to solubilize proteins – they are usually difficult to execute           coupled critical roles in DSB sensing, stabilization, signaling
and do not always yield positive results. Often poor solubility          and effector scaffolding: 1) expeditious establishment of
can be improved by changing a small number of amino acid.                protein-nucleic acid tethering scaffolds for the recognition
However, identifying which residues need to be changed can               and stabilization of DSBs; 2) initiation of DSB sensing,


cell cycle checkpoint signaling cascades, and establishment             5
of epigenetic marks via the ATM kinase; and 3) functional
regulation of chromatin remodeling at DSBs. Our results                 A hybrid approach to understanding virus structure and
suggest also how MRN mutations cause the human cancer                   evolution
predisposition diseases Nijmegen breakage syndrome and                  Sarah Butcher1, Juha Huiskonen1, Violeta Manole1, Nicola
ataxia telangiectasia-like disorder (ATLD). The combination             Abrescia2, Joseph Cockburn2, Jonathan Grimes2, Geoffrey
of solution and crystallographic structures with mutational             Sutton2, Jonathan Diprose2, Stephen Fuller2, Carmen San
and genetic analyses of dynamic DNA repair machinery                    Martin3, Roger Burnett3, David Stuart2, Dennis Bamford4,
provides paradigm-shifting information with implications                Jaana Bamford4
for prediction and intervention into biological and medical
                                                                         Institute of Biotechnology, University of Helsinki, P.O. Box
outcomes.                                                               65 (Viikinkaari 1), Helsinki, 00014 University of Helsinki,
                                                                        Finland, 2Division of Structural Biology, University of Oxford,
Hopfner, K.P., Karcher, A., Shin, D.S., Craig, L., Arthur,              The Wellcome Trust Centre for Human Genetics, Roosevelt
L.M., Carney, J.P., and J.A. Tainer. 2000. Rad50 ATPase:                Drive, Headington, Oxford, OX3 7BN, United Kingdom, 3The
ATP-driven control in DNA double-strand break repair & the              Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104,
ABC-ATPase superfamily, Cell 101: 789-800.                              4
                                                                         Institute of Biotechnology, University of Helsinki, P.O. Box
Williams, RS, Moncalian, G. Williams, JS, Yamada, Y,                    56, Helsinki, 00014 University of Helsinki, Finland
Limbo, O, Shin, DS, Groocock, LM, Cahill, D, Hitomi, C,                 Bacteriophage PRD1 is a prototypical virus with an
Guenther, G, Moiani, D, Carney, JP, Russell, P, Tainer JA               internal membrane. We have used a combination of X-ray
(2008) Mre11 Dimers Coordinate DNA End Bridging and                     crystallography, cryo-electron microscopy, genetics,
Nuclease Processing in Double Strand Break Repair, Cell                 biochemistry and comparative modelling to explore the
135: 97–109.                                                            structure and assembly of PRD1. X-ray crystallography of
Williams, R.S., Dodson, G.E., Limbo, O., Yamada, Y.,                    the virion allowed us to describe the structure and location of
Williams, J.S., Guenther, G., Classen, S., Glover, J.N.M.,              proteins P3, P16, P30 and P31. Virus assembly is controlled
Iwasaki, H., Russell, P., and Tainer, J.A. 2009. Nbs1 flexibly          by different structural proteins each with specialist roles. The
tethers Ctp1 and Mre11-Rad50 to coordinate Double-Strand                linearly extended P30 appears to nucleate the formation of the
Break Processing and Repair, Cell 139: 87-99.                           icosahedral facets (composed of trimers of the major capsid
                                                                        protein, P3) and acts as a molecular tape-measure, defining the
4                                                                       size of the virus and cementing the facets together. Pentamers
                                                                        of P31 form the vertex base, interlocking with subunits of
Dicer and Beyond: Making and Using RNA for Genetic                      P3 and the membrane protein P16. However, the structural
Control                                                                 organization of the components of the receptor-binding
Jennifer Doudna                                                         spike complex and the mediation of the different symmetry
MCB/Chemistry, UC Berkeley/HHMI, 708A Stanley Hall,                     mismatches between the spike-complex proteins was not clear.
Berkeley, CA, 94720                                                     We determined the architecture of the PRD1 spike complex
RNA molecules have been discovered at the heart of several              by using electron microscopy and three-dimensional image
aspects of gene expression, from protein biosynthesis by the            reconstruction of a series of PRD1 mutants. We constructed
ribosome to viral translation initiation to the targeting of new        an atomic model for the full-length P5 spike protein by using
proteins to the correct intracellular locale. Understanding how         comparative modeling. P5 was shown to bind directly to the
these RNA-mediated processes work will illuminate central               P31. P5 and the receptor-binding protein P2 form two separate
aspects of modern cell biology and also provide important               spikes, interacting with each other near the capsid shell. P5,
clues to the fundamental role of RNA in the evolution of                with a tumor necrosis factor-like head domain, may have been
life. I will describe our recent efforts to understand how the          responsible for host recognition before capture of the current
microRNA and siRNA processing machinery selects RNA                     receptor-binding protein P2. The architectural similarities of
substrates and how processed RNAs assemble with partner                 PRD1 with adenovirus and one of the largest known virus
proteins to form RNA-induced silencing complexes.                       particles, PBCV-1, support the idea that the mechanism of
                                                                        assembly of PRD1 is scaleable and applies across the major
                                                                        viral lineage formed by these viruses. Structural comparisons
                                                                        between bacteriophage PRD1 and adenovirus have revealed
                                                                        an evolutionary relationship that has contributed significantly
                                                                        to current ideas on virus phylogeny.


6                                                                         A hypothesis for the observed difference in stability behavior
                                                                          is that the trisulfide bond is more susceptible to thermally-
The organisation and dynamics of molecular chaperone                      induced bond cleavage than the corresponding disulfide.
complexes                                                                 This theory is supported by data showing a difference in
Johannes Buchner                                                          fragmentation rates observed in non-reducing, but not
Dept. Chemistry, Technische Universität München, 85747                    reducing, CE-SDS analysis.
Garching, Germany
Molecular chaperones are cellular machines for protein                    8
folding. They share the remarkable ability of selectively
recognizing and binding nonnative proteins. Many molecular                New and Evolving Analytical Methods for Evaluating
chaperones form large dynamic complexes. ATP binding and                  Protein Stability
hydrolysis allow most chaperones to shift between high-                   Alan Herman
and low-affinity states for binding substrate proteins. The               Analytical and Formulation Development, Althea
chemical energy is invested to generate an ordered sequence               Technologies, Inc., 5217 Verdugo Way, Ste. A, Camarillo,
of conformational changes in the chaperone protein which in               CA, 93012
turn drives binding and release in the substrate protein.                 Developing a stable protein formulation is dependent on
In the eukaryotic cell, Hsp90 with its cohort of co-chaperones            having good analytical technologies available for evaluating
is the most complex chaperone machinery. It contributes                   changes to the product. In order to fully understand
to the activation or conformational maturation steps of                   product stability it is necessary to evaluate biophysical,
hundreds of specific substrate proteins. This process is strictly         biochemical and biological changes to the product. Over
regulated at several levels. Intrinsically, the ATPase activity           the last 25 years our understanding of protein degradation
of Hsp90 is decelerated as a result of the slow conformational            and modification has increased significantly, as have our
changes leading to the hydrolysis-competent state. During                 analytical capabilities. Modern mass spectrometers give us
the chaperone cycle, several complexes are formed which                   the ability to get accurate masses on large intact proteins as
differ in the co-chaperones associated with Hsp90. Analysis               well as routine determination and localization of chemical
of these complexes revealed that co-chaperones bind to                    modifications such as deamidation and oxidation. Likewise,
specific conformational intermediates of Hsp90. This allows               our understanding of protein aggregation and self-association
inhibiting or accelerating the cycle at defined positions and it          has increased significantly in recent years. Using state-of-
sets the stage for the co-chaperone exchange required for the             the-art analytical methods such as field flow fractionation,
progression of the cycle.                                                 static and dynamic light scattering, and micro-flow imaging
                                                                          as well as more traditional methods including size exclusion
7                                                                         chromatography and analytical ultracentrifugation, we can
                                                                          accurately evaluate the degree, nature and rate of aggregation
Trisulfide Modification: Impact on Monoclonal Antibody                    in our formulations. This presentation will highlight several
Stability                                                                 case studies illustrating the application of these tools.
Karin Lucas
Protein Pharmaceutical Development, Biogen Idec, 5200                     9
Research Place, San Diego, CA, 92122
                                                                          Agitation-induced Aggregation of Monoclonal Antibodies
Trisulfide formation in monoclonal antibodies is a fairly                 and the Role of Container Closure Systems
recently discovered post-translational modification observed              Tia Estey
in biopharmaceutical products. This modification is caused                Protein Pharaceutical Development, Biogen Idec, 5200
by the addition of a sulfur atom across a disulfide bond to               Research Place, San Diego, CA, 92122
form a Cys-S-S-S-Cys linkage and is found predominantly
between the antibody heavy and light chains. Although                     Protein bipopharmaceuticals are exposed to mechanical
recent work suggests trisulfides do not impact conformation               agitation during manufacturing, fill-finish processing, and
or bioactivity, the effect of trisulfides on storage stability has        shipping and handling. Protein molecules are surface active
not been reported.                                                        and can accumulate and denature at air-liquid interfaces during
                                                                          mechanical agitation, leading to non-native aggregation. Non-
The stability of a model IgG1 antibody enriched in trisulfides            ionic surfactants (i.e. polysorbate 80) are routinely included
was compared to a preparation containing low trisulfide. At               in protein formulations to minimize surface-induced damage.
the intended long-term storage condition of 2-8 °C, similar               Polysorbate 80 was found to protect an agitation-sensitive
stability profiles were observed for the two materials.                   monoclonal antibody from the formation of insoluble and
However, under accelerated storage conditions, the trisulfide-            soluble aggregates during agitation studies, but the extent of
enriched material displayed more rapid rates of methionine                protection was found to be dependent on the surface area-
oxidation, soluble aggregate formation, and fragmentation.                to-volume ratio of the vial and fill volume. It was found


that low surface area-to-volume ratios were more damaging                   different environments to allow these opposing functions to
to the monoclonal antibody compared to the high surface                     exist within a single organelle.
area-to-volume ratios. A statistical model was developed to
determine the “worse case” conditions for agitation-induced                 12
damage. Optimization of the polysorbate 80 under the “worse
case” condition resulted in a robust formulation that is stable             NMR observation of structure, function and stability of
in a variety of drug product presentations.                                 proteins inside mammalian cells
                                                                            Masahiro Shirakawa
10                                                                          Department of Moecular Engineering, Graduate School of
                                                                            Engineering, Kyoto University, Kyoto-Daigaku Katsura,
Improving      physicochemical     properties     of                        Nishikyo-Ku, Kyoto, 615-8510, Japan
biopharmaceutical drug candidates                                           Due to high macromolecular contents, micro-space
Derrick Katatyama                                                           computerization, exposure of vast area of internal lipid
Amylin Pharmaceuticals, 9360 Towne Centre Drive, San                        membrane and other factors intra-cellular environments,
Diego, CA, 92121                                                            where most proteins work, are largely different from those
Often drug candidates in discovery and development                          of n vitro conditions we usually employ for biochemical and
have undesirable chemical and physical instability. This                    structural studies. Thus, it is possible that structure, function
presentation will focus on options for modifying the drug                   and stability of proteins are different between in vitro and in
molecules to improve their physicochemical properties such                  cells.
as chemical stability, solubility, and to lessen their potential for        In-cell NMR spectroscopy is applications of solution NMR
aggregation. Changes that will be discussed include creating                techniques to selectively observe proteins inside living cells.
analogs, chemical conjugation, and other modifications.                     Since established by Doetsch and co-workers, the method
                                                                            has been successfully applied to proteins over-expressed in
11                                                                          bacterial cells, as is exemplified by structure determination of
Protein folding in the ER: life and death decisions                         a protein in E. coli cells by Ito group in 2009. As its extension
Linda Hendershot, Ying Shen, Beata Lizak, Joel Otero,                       to eukatyotic cells, Selenko and we separately showed in-cell-
Yuki Okuda-Shimizu                                                          NMR spectra using oocytes from Xenopus laeis, to which
Genetics & Tumor Cell Biology, St. Jude Children’s Research                 isotope-labeled proteins were micro-injected
Hospital, 262 Danny Thomas Pl, Memphis, TN, 38112                           We established a method to analyse proteins in human
The endoplasmic reticulum is the site of synthesis of nearly all            HeLa cells by usnf 1H-15N correlation two-dimensional
proteins expressed on the cell surface or secreted. They enter              NMR spectroscopy. The proteins were delivered to cells by
the ER through a protein channel as extended polypeptide                    the action of cell-penetrating peptide (CPP) linked to the
chains, which interact with a variety of molecular chaperones               proteins. NMR and fluorescence microscopy observations
and folding enzymes that help them achieve their proper                     suggest that the detachment of CPP inside cells is required
mature tertiary and quaternary structures and allow them to                 for acquisition of high quality NMR spectra and smooth
traffick further along the secretory pathway. If however, the               diffusion of transduced proteins.
nascent protein fails to fold and assemble properly, it must be             The in-cell NMR spectroscopy provides information
identified and targeted for retrotranslocation through another              about structure, interaction and stability of proteins under
protein channel for degradation by the 26S proteasome,                      intracellular environment. The spectral comparison between
which involves some of the same molecular chaperones.                       wild—type ubiquitin and its mutant suggests that wild-
How the ER can distinguish between proteins that have not                   type ubiquitin makes interactions with endogenous cellular
yet folded and those that cannot fold is not clear, as it is                proteins. The in-cell NMR spectra of FKBP12 showed that
expected that both types of proteins would have very similar                immunosuppressants, FK506 and rapamycine, administered
features. In addition, the folding of many nascent proteins is              to extracellular fluid enter the cells and bind to the protein.
dependent on the oxidizing environment of the ER, whereas                   Thus, the method can potentially be a useful step in drug
the degradation of misfolded proteins often requires that                   development processes. In-cell NMR and related method
those regions that have folded be reduced to allow them to                  revealed that the exchange rates of most highly protected
pass through the retrotranslocon. Recent data suggests that                 main chain amide hydrogens of ubiquitin are markedly higher
molecular chaperones might be able to participate in both                   in cells than in vitro. This observation raises a possibility that
protein folding and degradation through the association of                  the protein is unstabilized in cells.
distinct subsets of co-chaperones. In addition, the finding
that folding and degradation might be spatially separated
in the ER provides a possible mechanism for establishing


13                                                                    of protein structure prediction, whose possibility appeared
                                                                      guaranteed in principle by the thermodynamic hypothesis.
Putting molecules in context with correlated fluorescence             So, I stayed for three years at Professor Scheraga’s lab where
and x-ray tomography                                                  this problem was being attacked computationally. My three
Carolyn Larabell1, Mark Le Gros2                                      years were spent mainly for development of computational
  Anatomy, University of California, San Francisco, 1550 4th          methods to treat conformational fluctuations.
St., Box 2722, Rock Hall, Mission Bay, San Francisco, CA,
94158, 2Physical Biosciences, Lawrence Berkeley National              Because I could get a job of Associate Professor in 1971 at
Laboratory, 1 Cyclotron Rd., MS6-2100, Berkeley, CA,                  Kyushu University in Japan, I started a new challenge of
94720                                                                 theoretical and computational study of protein folding process
                                                                      with an idea of elucidating the basis of prediction. Statistical
Soft X-ray tomography is a high throughput, high fidelity             mechanics of phase transition is a big field in theoretical
imaging technology that examines whole, hydrated eukaryotic           physics. I wished to construct a statistical mechanics of
cells in three-dimensions at 20-50 nm resolution. Cells are           order-disorder transition in a system whose ordered state
imaged at x-ray energies between the K shell absorption edges         is the specific three-dimensional protein structure dictated
of carbon (284 eV, 4.4 nm) and oxygen (543 eV, 2.3 nm),               by the amino acid sequence. With anticipation that the
where photons readily penetrate the aqueous environment               essential character of such a transition is common to any
while encountering significant absorption from carbon- and            highly specific ordered state, I devised for extracting such an
nitrogen-containing organic material. Because organic                 essence a drastically simplified theoretical model – a lattice
material absorbs approximately an order of magnitude                  model of protein with highly specific interactions1. Various
more strongly than water at these energies, we obtain a               variations of such a model are now widely used for studying
quantifiable natural contrast image of cellular structures.           protein folding as well as even function. By incorporating
Our soft x-ray cryo-tomography stage enables rotation of the          various intramolecular forces operative in real proteins
specimen through more than 180 degrees, making it possible            into the simplified model and by carrying out simulation of
to collect multiple images of the entire specimen. We then            folding process, we learned many things. Summarizing such
use tomographic reconstruction procedures to generate high            results, I wrote a review2 in 1983, in which I advocated what
contrast, 3-D views of the cells at isotropic resolution. We          I called “the consistency principle.” This is an assertion that
obtain statistically significant information because large            various intramolecular forces operative in the native protein
numbers of cells can be imaged in a short period of time (150         structure are consistent with each other. After a similar idea
msec per image). Because the cells have not been processed            was presented by Wolynes3 in 1987 under the name of the
(chemically fixed, dehydrated, embedded, sectioned, stained,          principle of minimum frustration, the idea appeared generally
etc.) as is required for most other high resolution imaging           accepted as a general principle. The principle postulates that
technologies, the information obtained accurately reflects            amino acid sequences satisfying the condition of consistency
the native state of the cells. To determine the positions of          have been selected during the process of molecular evolution.
molecules with respect to cellular structures, we developed a         In the lecture I shall explore how the consistency principle
novel cryogenic light microscope that collects high resolution        together with the fact of marginal stability of native proteins
fluorescent images of the vitrified sample prior to performing        accounts for various aspects of protein folding. The drastically
soft x-ray tomography. This multi-modal approach allows               simplified model I used for simulation of protein folding is an
labelled molecules to be localized in the context of a high-          expression of the consistency principle as a theoretical model.
resolution 3-D tomographic reconstruction of the cell. I will         From the study aiming at developing prediction method, a
describe these technologies and provide examples of recent            general principle was discovered.
applications to biomedical research projects.
                                                                      How about the ambition of developing a method of protein
                                                                      structure prediction? Parallel with the efforts of elucidating
                                                                      the basis of prediction, I was trying various ideas. One is a
Seeking general views to look at proteins                             combination of experimental information and computational
Nobuhiro Go                                                           prediction. Especially I was interested in NOE information
RIKEN Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun,              that can be obtained by NMR experiment. Together with W.
Hyogo, 679-5148, Japan                                                Braun, I discovered that NOE information alone can determine
Nirenberg’s first paper on genetic code was published in              protein structures to the atomic resolution4. The computational
1961, just at the time when I started my graduate study in            method for deducing protein three-dimensional structures
particle physics in Tokyo University. Then, Anfinsen’s                from experimental data was used by K. Wuthrich. Thus, a
refolding experiment and the idea of the thermodynamic                new experimental method of protein structure determination
hypothesis were conveyed. Stimulated by these big historical          was born. I regard the most important here is the discovery of
events, I changed my major. Wishing to take an advantage              the fact NOE data contain enough information to determine
of my physics background, I wanted to tackle the problem              atomic resolution protein structures. Once this is known,


algorithm development is not difficult. I felt deeply satisfied         At least one hydrogen-bond has to be separated in the
for having contributed to the birth of the new methodology.             channel for stopping proton permeation, but the separation
As in the study of prediction method, my attention was always           increases energy barrier reducing the water permeation
directed to conformational fluctuation. From around 1980,               speed. For answering such puzzling question, structure of
I began studying conformational dynamics in the folded                  AQP1 was analyzed [1] by an originally developed cryo-
native state and related biological function. At that time there        electron microscope [2]. The analysis revealed characteristic
were influential papers reporting highly non-linear complex             structural determinants including an unusual fold. We
behavior of native state dynamics. Nonetheless, I dared to              proposed H-bond isolation mechanism for understanding
develop a linear normal mode analysis5,6, believing that, in            the proton exclusion mechanism keeping high speed water
any complex system, the first step to understand it is to find a        permeation. Structure analysis of AQP0 at a resolution of 1.9
linear aspect in it. This was a choice I learned from my study          Å shows clear densities of water molecules in the channel [3],
of physics. I think correctness of this choice has been proven          but just 3 water molecules were observed at the closed state.
by the fact that the normal mode analysis is now used very              By improving the resolution of the first structure of AQP4
widely as a general tool to study protein dynamics. I also              [4], 8 water molecules were discriminated in the channel and
have applied to many problems.                                          we confirmed the H-bond isolation mechanism based on the
                                                                        structure analysis [5]. We confirmed that AQP4 molecule
Upon reflection, I now realize that I have been seeking general         acquired two functions of cell adhesive and channel functions
views to look at proteins.                                              and named this type channel “Adhennel” by concatenating
After I moved to Kyoto University in 1987 as Professor,                 two words of adhesive and channel [4]. Structure of typical
I became deeply involved in national and international                  Adhennel, connexin 26 (Cx26) was analyzed by electron
administrative duties, while watching bright students in the            and X-ray crystallography and a plug gating mechanism was
lab. One of such an effort is to run a rather large national            proposed [6, 7]. (The source of research support: NEDO)
research consortium in protein physico-chemistry. This                  [references]
consortium became one of the movements toward the
establishment of Protein Science Society of Japan, which is             1 K. Murata et al., Nature 407, 599-605 (2000).
one of the supporting body of this Protein Society.                     2 Fujiyoshi Y., Adv. Biophys. 35, 25-80 (1998).
After resignation from all administrative duties about                  3 T. Gonen et al., Nature 438, 633-638 (2005).
three years ago, I started a new challenge of developing an             4 Y. Hiroaki et al. J. Mol. Biol. 355, 628-639 (2006).
algorithm of single molecule imaging by X-ray free electron
laser.                                                                  5 K. Tani et al. J. Mol. Biol. 389, 694-706 (2009).

Taketomi, H. et al. (1975) Int.J. PeptideProtein Res. 7, 445-           6 A. Oshima et al. PNAS 104, 10034-10039 (2007).
459.                                                                    7 S. Maeda et al., Nature 458, 597-602 (2009).
Go, N. (1983) Ann. Rev. Biophys. Bioeng. 12, 183-210.
Bryngelson, J.D., Wolynes, P.G. (1987) Proc. Natl. Acad. Sci.
USA 84, 7524-7528.                                                      Structure, function, and inhibition of human O-GlcNAc
Braun, W., Go, N. (1985) J. Mol. Biol. 186, 611-626.                    transferase
                                                                        Suzanne Walker
Noguti, T., Go, N. (1982) Nature 296, 776-778.                          Microbiology and Molecular Genetics, Harvard Medical
Go, N. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 3696-               School, 200 Longwood Avenue, Boston, MA, 02115
3700.                                                                   O-GlcNAc transferase is an essential mammalian enzyme that
                                                                        functions as a nutrient sensor to couple metabolic status to the
15                                                                      regulation of a wide variety of cellular signaling pathways.
Structural     Physiology     Based      on      Electron               OGT catalyzes the transfer of N-acetyl glucosamine from
Crystallography                                                         UDP-GclNAC to serines and threonines of cytoplasmic,
Yoshinori Fujiyoshi                                                     nuclear, and mitochondrial proteins, including transcription
Graduate School of Science, Kyoto University, Oiwake-cho,               factors, tumor suppressors, kinases, phosphatases, and
Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan                         histone modifying proteins. Aberrant O-GlcNAcylation by
                                                                        OGT has been directly linked to insulin resistance, diabetic
Water permeation through biomembranes is strictly separated             complications, cancer and neurodegenerative diseases
from the movement of ions. For example, aquaporin-1                     including Alzheimer’s. We will report the crystal structure
(AQP1) was confirmed to exclude any ions, solutes and                   of human OGT, both as a binary complex with UDP and as
even proton keeping effective water permeation. Water                   a ternary complex with a peptide substrate, and will discuss
molecules form hydrogen-bond chain through which protons                the mechanistic implications of the structures. We will also
can be permeated as domino toppling with great efficiency.


describe progress towards the development of inhibitors                  high affinity, and 2) a strategy that can be applied to generate
useful as cellular probes to explore the biology of OGT and              other dual specific antibodies toward two defined targets. The
to assess its potential as a therapeutic target.                         application of the strategy to generate a therapeutic candidate
                                                                         will also be discussed.
Stapled α-Helical Peptides: The Twists and Turns of Drug
Discovery                                                                Engineering ultra-stable molecular recognition proteins
Tomi Sawyer                                                              using the Top7 protein scaffold
Aileron Therapeutics, Inc., 840 Memorial Drive, Cambridge,               Cheryl Baird, Curt Boschek, Garry Buchko, Kristin Victry
MA, 02139                                                                Pacific Northwest National Lab, 902 Battelle Blvd, PO Box
Intracellular protein-protein interactions frequently involve            999, Richland, WA, 99352
helical peptide molecular recognition to modulate signal                 In the last decade, new classes of molecular recognition
transduction, proliferation, survival, metabolism, transcription,        proteins (MRP) based on small, robust scaffolds have
translation and other regulatory pathways. Amongst a plethora            emerged as an alternative to immunoglobulin-based MRPs.
of potential therapeutic targets and pathways involving                  These novel MRP scaffolds could be utilized as diagnostic
such helical peptide molecular recognition are BH3:Mcl-1,                reagents, basic research tools, or therapeutics, and may offer
p53:hDM2/X, and MAML-1:Notch. Noteworthy, the BH3-                       potential advantages in production and application over
only subfamily of apoptotic modulatory proteins has provided             conventional immunoglobulin-based MRPs. We have been
the inspiration for the first in vivo effective synthetic peptide        developing one such scaffold, Top7, as an MRP for diagnostic
incorporating a macrocyclic hydrocarbon staple to stabilize              and basic research applications. This small, synthetic protein
helical conformation and confer metabolic stability, cell                is thermodynamically stable, and can be easily engineered
penetration, and outstanding pharmacokinetic properties                  and produced in microbial systems.
(Walensky et al., Science [2004]). In this presentation, the             We have previously shown that insertion of a 12-residue
design, structure-property and in vivo efficacy of a second-             binding element (CB1) confers specific binding of Top7 to the
generation of promising BH3 stapled helical peptides will                CB1 target (CD4) without affecting the scaffold’s stability.
be described with respect to identifying a clinical candidate            Since multiple binding elements improve the specificity and
for the treatment of Bcl2-dysregulated cancers. Comparative              affinity of MRPs, we have identified 4 additional loops in
analysis of BH3, p53 and MAML-1 stapled helical peptides                 Top7 that are not involved in the protein’s secondary and
will be further described relative to key in vitro and in vivo           tertiary structures and should accommodate insertions without
structure-property relationships. Ultimately, the development            compromising the scaffold’s stability. We have developed
of a new therapeutic modality is envisaged to leverage the               variants of Top7 with the CB1 element inserted into these
unique drug properties of stapled helical peptides.                      loop regions to determine whether these sites are available
                                                                         for target binding without affecting the stability of Top7.
18                                                                       The circular dichroism spectra of the Top7-CB1 variants is
Two-in-One antibody that interacts with two different                    the same as that of native Top7, suggesting that insertion
molecules at the antigen-binding site with high affinity                 into these loops does not alter the core Top7 scaffold. The
Germaine Fuh                                                             denaturation midpoints of the Top7-CB1 variants in guanidine
Antibody Engineering, Genentech, 1 DNA way, south San                    hydrochloride are also similar to that of native Top7 (5-6M).
Francisco, CA, 94080                                                     All four Top7 variants were able to specifically detect CD4
                                                                         by ELISA. These experiments suggest that insertion into any
We explore the ability of one antigen-binding site to interact           one of these loops does not affect the stability of Top7.
with two unrelated protein antigens by an engineering
approach to evolve a mono-specific antibody into dual                    We are currently constructing libraries of naïve human
specific antibodies. By mutating the light chain CDRs of                 complementarity determining region insertions into these 4
Herceptin, we generate a repertoire of antibody variants and             locations in Top7. These libraries will provide the foundation
isolate the variants that not only retain HER2 binding but also          for an ultra-stable MRP that can be rapidly engineered in vitro
bind to secondary antigens. Crystallographic and mutagenesis             for applications in diagnostics and field-based bio-detectors.
studies of a HER2/VEGF dual specific Fab revealed an
extensive overlap of the antibody surface areas contacting
the two antigens, but distinct sets of residues are shown to
energetically engaged with the two antigens. The high dual
affinity is achieved and translated to dual action in vitro and
in vivo. The results demonstrate 1) the capability of antibody-
combining site to interact with two unrelated antigens with


20                                                                       and necessitating the development of an entirely new set
                                                                         of experimental and analytical techniques. The importance
Delivering antibody therapeutics to where the action is-                 of developing new methodology to study these proteins is
inside the cell                                                          underlined by the fact that IDPs are associated with many
Anthony Kossiakoff, Shahir Rizk, Crista Brawley, Serdar                  human diseases, including cancer, cardiovascular disease,
Uysal, Ronald Rock                                                       amyloidosis, neurodegenerative disease and diabetes.
Biochemistry and Molecular Biology, University of Chicago,
Chicago, IL, 60637                                                       In order to describe the conformational behaviour of IDPs, a
                                                                         molecular representation of the disordered state is required,
Monoclonal antibody therapy has emerged as a major                       based on diverse sources of structural data that often exhibit
therapeutic modality for use against cancer. A critical                  complex and very different averaging behaviour. The high
limitation is that antibodies can only be directed at                    intrinsic flexibility of IDPs makes ensemble descriptions
extracellular targets because they cannot penetrate the                  particularly appropriate to describe the conformational
cell membrane. This eliminates a rich set of potential                   equilibrium sampled by the protein in solution. We have
cytoplasmic targets that function as crucial components of               developed approaches to describe representative structural
signaling pathways or structural networks. To overcome this              ensembles of IDPs directly from the experimental NMR and
limitation we have developed a receptor-mediated delivery                small angle scattering data. We have initially applied these
system that has proven effective in internalizing a novel class          techniques in conjunction with residual dipolar couplings,1-4
of synthetic antibodies in their fully functional form. An               demonstrating for example, site specific mapping of
important attribute of this delivery system is that it inherently        conformational space in intrinsically disordered viral proteins,
seeks out certain types of cancer cells, while being inert               in p53 and tau protein and in urea denatured ubiquitin.
towards normal tissue. In contrast to traditional antibodies,            We have also incorporated the use of MTSL-induced
our synthetic antibodies (sABs) are generated in vitro using a           paramagnetic relaxation enhancements in α-synuclein. More
phage display methodology. Combined with various sorting                 recently we have shown that in combination with 13C and
strategies, sABs can be engineered with unique properties                15
                                                                           N chemical shifts alone we can identify entire secondary
such as: 1) targeting a specific region of a protein surface;            structural elements and their associated populations, as well
2) recognizing a specific conformational or oligomeric                   as characterizing the subtle detail of local conformational
state; 3) inducing a conformational form and 4) blocking                 sampling in unfolded proteins. This raises the exciting
a specific protein-protein interaction. As a demonstration               prospect of probing the conformational behavior of IDPs
of the power of this approach, we have generated a set of                under conditions where additional parameters cannot be
sABs tailored to induce conformational changes in F-actin                easily measured but where chemical shifts are still readily
that substantially alter the actin cytoskeleton by mechanisms            available, for example in crowded or cellular environments
involving depolymerizing, severing, bundling and capping of
the actin filaments. We introduce these sABs into cancer cells           1. Salmon L., Nodet G., Ozenne V., Jensen M.R., Zweckstetter
using our receptor-mediated delivery system and show that                   M. and Blackledge M., J. Am. Chem. Soc., In press.
properties selected for during in vitro phage display sorting            2. Jensen M.R., Salmon L., Nodet G. and Blackledge M., J.
are accurately recapitulated in live cells. The effects of actin            Am. Chem. Soc., 132, 1270-1272 (2010)
filament bundling are shown to be particularly detrimental               3. Jensen M.R., Markwick P.R., Meier S., Griesinger C.,
to the cancer cells, suggesting that sABs performing this                   Zweckstetter M., Grzesiek S., Bernado P. and Blackledge
function may be useful as therapeutic agents.                               M., Structure, 17, 1169-1185 (2009)
                                                                         4. Nodet G., Salmon L., Ozenne V., Meier S., Jensen M.R.
21                                                                          and Blackledge M., J. Am. Chem. Soc., 131, 17908-17918
Relating Primary Sequence to Protein Function in                            (2009)
Intrinsically Disordered Proteins by NMR                                 5. Bernado, P.; Blanchard, L.; Timmins, P.; Marion, D.;
Loic Salmon, Valery Ozenne, Guillaume Communie, Luca                        Ruigrok, R. W. H.; Blackledge, M. Proc. Natl. Acad. Sci.
Mollica, Malene Jensen, Martin Blackledge                                   U.S.A. 2005, 102, 17002–17007.
Institute de Biologie Structurale, 41 rue Jules Horowitz,                6. Wells, M.; Tidow, H.; Rutherford, T. J.; Markwick, P.;
grenoble, France                                                            Jensen, M. R.; Mylonas, E.; Svergun, D.I.; Blackledge,
The realisation that a large fraction of functional proteins                M.; Fersht, A.R. Proc. Natl. Acad. Sci. (U.S.A.) 2008, 105,
encoded by the human genome are intrinsically disordered or                 5762–5767
contain long disordered regions has revealed a fundamental               7. Jensen M.R., Houben K., Lescop E., Blanchard L., Ruigrok
limitation of classical structural biology. Intrinsically                   R.W.H. and Blackledge M., J. Am. Chem. Soc., 130, 8055-
disordered proteins (IDPs) are functional despite the lack of               8061 (2008)
well-defined structure, imposing a new perspective on the
relationship between primary protein sequence and function,


22                                                                    the unfolded chain. The observation of a collapse of similar
                                                                      extent in the extremely hydrophilic, intrinsically disordered
Structural Reorganization of α-Synuclein With pH                      protein prothymosin α suggests that the hydrophobic effect
Observed by Replica Exchange Molecular Dynamics                       is not the sole source of the underlying interactions. Circular
(REMD) Simulations and Nulear Magnetic Resonance                      dichroism spectroscopy and replica exchange molecular
(NMR)                                                                 dynamics simulations in explicit water show changes in
Ronald Levy1,2, Chitra Narayanan1,3, Michael Andrec1,2,               secondary structure content with increasing temperature and
Daniel Weinstock1, Kuen-Phon Wu1, Jean Baum1,2                        suggest a contribution of intramolecular hydrogen bonding to
  BioMaPS Institute for Quantitative Biology, 2Chemistry              unfolded state collapse.
& Chemical Biology, 3Biochemistry, Rutgers, The State
University of New Jersey, 610 Taylor Road, Piscataway, NJ,            Intrinsically disordered proteins (IDPs) often contain a large
08854                                                                 fraction of charged amino acids. We find that, in contrast to
                                                                      the compact unfolded conformations that have been observed
Intrinsically disordered proteins (IDPs) are disordered under         for many proteins at low denaturant concentration, IDPs
physiological conditions with the propensity to remain                can exhibit a prominent expansion at low ionic strength
disordered being encoded in the amino acid sequence.                  that correlates with their net charge. Charge-balanced
α-Synuclein , implicated in Parkinson’s disease, is one such          polypeptides, however, can exhibit an additional collapse
IDP, whose aggregation rate and the rate of conversion from           at low ionic strength, as predicted by polyampholyte theory
monomer to aggregate, are sensitive to changes in amino acid          from the attraction between opposite charges in the chain. The
sequence and environmental conditions. Using a combination            pronounced effect of charges on the dimensions of unfolded
of REMD Simulations and NMR, we show how a shift in                   proteins has important implications for the cellular functions
pH which alters the charge distribution in α-Synuclein,               of IDPs.
reorganizes the α-Synuclein structural ensemble. From
simulations of α-Synuclein in implicit solvent over a range           24
of thermodynamic conditions, we characterize the polymeric
behavior of the polypeptide chain and compare the results with        Structural disorder and aberrant proteins in the cell
simplified polymer models. I will also present our approach           Peter Tompa
for using REMD simulations to interpret NMR Residual                  Institute of Enzymology, Biological Research Center,
Dipolar Coupling Patterns for α-Synuclein and other IDPs.             Hunagrian Academy of Sciences, Budapest, Hungary
This work has been supported by the National Institutes of            Structural disorder is widespread in eukaryotic organisms
Health grants GM45302 and GM30580.                                    and it correlates with important regulatory functions, due to
                                                                      which it is often involved in diseases such as cancer. Here
23                                                                    we carried out systematic bioinformatic studies to address if
Probing interactions in unfolded proteins with single                 structural disorder confers selective advantages on proteins
molecule fluorescence spectroscopy                                    that arise suddenly and unexpectedly in the cell, either due
Benjamin Schuler                                                      to chromosomal translocations [1] or dual-coding elicited
Biochemisches Institut, Universität Zürich,Winterthurerstrasse        by alternative splicing [2]. The former often gives rise to
190, Zurich, 8057, Switzerland                                        chimeric proteins by fusing segments of two distinct genes
                                                                      in cancer, whereas the latter generates novel protein products
We used single molecule Förster resonance energy transfer             translated in alternative reading frames under physiologic
(FRET) in combination with other biophysical methods to               conditions. We have selected 406 translocation-related human
investigate the effect of temperature and charge interactions         proteins and 99 human genes with alternative splice variants
on the dimensions of unfolded proteins. With single molecule          comprising a dual-coding region at least 75 nucleotides in
FRET, this question can be addressed even under near-native           length. Translocation-related human proteins are significantly
conditions, where most molecules are folded, allowing                 enriched in disorder (43.3% vs. 20.7% in all human proteins),
us to probe a wide range of denaturant concentrations and             which is particularly conspicuous at the vicinity of the
temperatures.                                                         breakpoint. In the case of dual coding, structural disorder is
We find a compaction of the unfolded state of a small                 most apparent in -1 frameshifts, where the average disorder
cold shock protein with increasing temperature both in the            increases from 22.2% in the ancestral frame to 58.2% in the
presence and absence of denaturant, with good agreement               derived frame. Our results suggest that structural disorder
between the results from single molecule FRET and dynamic             enables these aberrant protein products to exist without the
light scattering. While dissociation of denaturant from the           need of a highly evolved three-dimensional fold that would
polypeptide chain with increasing temperature accounts for            require a lengthy selection procedure. Thus, structural disorder
part of the compaction, the results indicate an important role        in both cases enables effective mechanisms to operate in the
for additional temperature-dependent interactions within              sudden evolutionary appearance of novel protein functions,
                                                                      let they be pathologic or physiologic in nature.


1. Hegyi, H., L. Buday, and P. Tompa (2009) Intrinsic                   a critical force Fc. By solving the Langevin equation under
   structural disorder confers cellular viability on oncogenic          conditions of a force-quench we generate folding trajectories
   fusion proteins. PLoS Comput Biol 5: e1000552.                       corresponding to the diffusional collapse of an extended
2. Kovacs, E., P. Tompa, K. Liliom, and L. Kalmar (2010)                protein. The resulting trajectories reproduce, uncannily, the
   Dual coding in alternative reading frames correlates with            different stages as well as the magnitude and time course of
   intrinsic protein disorder. Proc Natl Acad Sci U S A 107:            the collapse trajectories observed experimentally in single
   5429-34.                                                             proteins. Our studies challenge the simplified views of
                                                                        protein folding that emerged from bulk experiments, while
25                                                                      providing novel benchmarks to uncover the physics of protein
                                                                        dynamics, essential to understand biology.
Protein folding/unfolding under force
Julio Fernandez1, Ronen Berkovich2, Tzu-Ling Kuo3, Sergi                26
Garcia-Manyes4, Jingyuan Li5, Itay Barel2, Hui Lu6, Bruce
Berne7, Michael Urbakh8, Joseph Klafter9                                Proteins under tension: insights from single-molecule
  Biological Sciences, Columbia University, 1212 Amsterdam              force spectroscopy
Avenue, New York, NY, 10027, 2School of Chemistry, Tel                  Gerhard Hummer
Aviv University, Room 218, Ornstein Building, School                    Laboratory of Chemical Physics, National Institutes of
of Chemistry, Tel Aviv University, Tel Aviv, 69978, Israel,             Health, Bldg. 5, Rm. 132, Bethesda, MD, 20892-0520
  Physics, Columbia University, 538 West 120th St, mail code            Single-molecule force spectroscopy using laser tweezers and
9319, New York, NY, 10027, 4Biological Sciences, Columbia               atomic force microscopes provides an exceedingly detailed
University, 1011C Fairchild Center, 1212 Amsterdam                      picture of the mechanical properties of individual molecules
Avenue, New York, NY, 10027, 5Chemistry, Columbia                       and molecular assemblies. By exerting mechanical force on
University, 3000 Broadway, mail code 3159, New York, NY,                trapped molecules, molecular transitions can be monitored
10027, 6Bioengineering, University of Illinois at Chicago,              with sub-nanometer positional resolution, including the
Room 218, 851S Morgan St, Chicago, IL, 60607, 7Chemistry,               folding and unfolding of individual proteins and nucleic acids,
Columbia University, 3000 Broadway, mail code 3103, New                 and the dissociation of molecular complexes. In my talk, I
York, NY, 10027, 8School of Chemistry, Tel Aviv University,             will give an overview of the theory of single-molecule force
Room 208, Ornstein Building, School of Chemistry, Tel Aviv              spectroscopy. In particular, I will show how single-molecule
University, Tel Aviv, 69978, Israel, 9School of Chemistry, Tel          pulling can be used to extract free energies and kinetic rate
Aviv University, Room 209, Ornstein Building, School of                 coefficients of molecular processes. I will also show how
Chemistry, Tel Aviv University, Tel Aviv, 69978, Israel                 molecular simulations help in the analysis and interpretation
Single molecule force-clamp spectroscopy has opened up new              of force measurements of protein folding.
approaches to study the protein folding/unfolding reaction at
the single molecule level. The single molecule data departs             27
substantially from the simplified two-state folding/unfolding           A Tangled Problem: the Structure, Function and Folding
behavior that is commonly observed in bulk. For example,                of Knotted Proteins
under constant force conditions single ubiquitin proteins               Sophie Jackson1, Anna Mallam1,2, Fredrik Andersson1, Danny
show non-exponential unfolding kinetics which indicates the             Hsu1, Elizabeth Werrell1, Joe Rogers1, Lindsay McMorran1,3,
presence of static disorder in the activation energy barrier for        Liz Morris1,4, Will Crone1
unfolding. The measured variance in ΔE shows both force                 1
                                                                         Chemistry Department, Cambridge University, Lensfield
dependent and independent components, where the force                   Road, Cambridge, CB2 1EW, United Kingdom, 2Institute
dependent component scales with F2, in excellent agreement              for Cellular and Molecular Biology, University of Texas,
with static disorder theory. Extended proteins folding after an         1 University Station, Austin, TX, TX 78712, 3Institute of
abrupt quench in the pulling force follow one of a multitude            Molecular and Cellular Biology, Leeds University, Astbury
of possible trajectories, where each trajectory reflects a              Centre, Leeds, LS2 9JT, United Kingdom, 4Centre for
distinct conformational ensemble. The observed trajectories             Biomolecular Sciences, Nottingham University, University
are the result of diffusion along the free energy of the protein        Park, Nottingham, NG7 2RD, United Kingdom
over the end-to-end length coordinate. This is readily
demonstrated by modeling the free energy of the protein as              Since 2000, when they were first identified by Willie Taylor,
a combination of an entropic elasticity term together with              the number of knotted proteins within the pdb has increased
a short range potential representing enthalpic hydrophobic              and there are now nearly 300 such structures. The polypeptide
interactions. The resulting free energy of the molecule shows           chain of these proteins forms a topologically knotted structure.
a force dependent energy barrier of magnitude ΔE=ε(F- Fc)3/2
                                                                       There are now examples of proteins which form simple 3-1
separating the enthalpic and entropic minima that vanishes at           trefoil knots, 4-1, 5-2 Gordian knots and 6-1 Stevedore
                                                                        knots. Recent AFM pulling experiments established that the


mechanical unfolding of such structures does not result in a           28
linear chain but it a state in which the polypeptide chain is
still knotted.                                                         Protein Folding Dynamics on and off the Ribosome
                                                                       Silvia Cavagnero
Knotted proteins represent a significant challenge to both the         Chemistry, University of Wisconsin Madison, 1101 University
experimental and computational protein folding communities.            Ave., Madison, WI, 53706
When and how the polypeptide chain knots during the folding
of the protein poses an additional complexity to the folding           The pathways of protein folding in vivo are still poorly
landscape and recent computational studies have shown that             understood despite the tremendous impact of correct folding
folding is very inefficient when the standard Go-type models           for cell function and disease prevention. In the case of small/
are applied to these knotted systems.                                  medium-size proteins, kinetic arguments suggest there is
                                                                       ample time for cotranslational conformational sampling,
We have been studying the structure, folding and function              while nascent chains are still ribosome-bound and before
of two types of knotted proteins – the 3-1-trefoil knotted             synthesis of the full length protein is complete. Given the
methyltransferases and 5-2-knotted ubiquitin C-terminal                complexity of the cellular environment, a combination of
hydrolases. The talk will focus on the work we have done               tailored biological and spectroscopic tools is likely needed
on two knotted methyltransferases - YibK and YbeA. We                  to drive progress in this area. This lecture will present our
established some years ago, that these two proteins could              current efforts to understand how proteins acquire the ability
be reversibly unfolded in vitro using chemical denaturants             to independently fold as nascent chains elongate from N to
and that the equilibrium unfolding of these dimeric structures         C terminus. The role of the ribosome in modulating nascent
was very similar to that of other dimeric proteins of the same         protein dynamics, and the structural rearrangements that take
size. Detailed kinetic studies were then used to show that             place upon release of fully synthesized nascent proteins from
the unfolding/folding pathway of YibK was complex with                 the ribosome will also be discussed.
multiple intermediate states populated and parallel pathways
which at that time were assigned to proline isomerisation
events. A late intermediate on the folding pathway was
shown to be monomeric and similar to that populated during             Lactose Permease
equilibrium unfolding experiments, as well as a partially              Howard Kaback
unfolded monomeric form which could be formed by                       Physiology, University of California, Los Angeles, MRL Rm.
lowering the pH or destabilising the dimer interface.                  6-720, 675 Charles E. Young Drive South, Los Angeles, CA,
We have used protein engineering techniques on both small              90095
and large scale to probe the knotting and folding of both              Lactose permease (LacY) catalyzes the coupled, stoichiometric
YibK and YbeA. Fusions of the two methyltransferases with              translocation of a galactopyranoside and an H+ across the
the stable ThiS domain at both N- and C-termini were used              cytoplasmic membrane of Escherichia coli. X-ray crystal
to create proteins with the deepest knots known to date. A             structures have been solved in an inward-facing conformation
kinetic analysis of these fusions generated some unexpected            and confirm many conclusions from biochemical and
results and demonstrated that knotting was likely to occur in          biophysical studies. LacY contains N- and C-terminal
a highly unstructured state, possibly even the denatured state.        domains, each with 6 largely irregular transmembrane
In contrast, protein engineering analysis of highly conserved          helices that are positioned pseudo-symmetrically. The
residues located in the knotted region and thought to stabilise        helices surround a large hydrophilic cavity exposed to the
the knotted structure only affected the later stages of folding        cytoplasm. The sugar-binding site is located at the two-fold
suggesting that the interactions of these side chains was not          axis of pseudo-symmetry at the apex of the cavity in the
formed early and therefore unlikely to play a role in initial          approximate middle of the molecule. Access to the sugar-
threading events. Mutation of cis Proline also demonstrated            binding site from the periplasmic side is completely blocked.
that the parallel pathways observed in the folding of YibK             The residues involved in sugar binding and H+ translocation
were not attributable to proline isomerisation events.                 have been identified, and surprisingly, those involved in H+
Very recently, we have made constructs of YibK and                     translocation are aligned parallel to the membrane at the same
YbeA which contain cysteine residues at N- and C-termini               level as the sugar-binding site. Both sites may be exposed
and we have used disulphide bond formation to probe the                alternatively to water-filled cavities in the inward- or the
conformation of the polypeptide chain in the denatured                 outward-facing conformation, thereby allowing sugar and H+
state. Remarkably, we have shown that both YibK and YbeA               release from either side of LacY. These features may explain
adopt a knotted conformation in their highly unstructured,             how LacY catalyzes lactose/H+ symport in both directions
chemically unfolded states.                                            across the membrane utilizing the same residues. Site-directed
                                                                       alkylation, single molecule fluorescence energy transfer
                                                                       (smFRET), double electron electron resonance (DEER) and
                                                                       thiol cross-linking studies all support a mechanism involving


alternating access of both the sugar- and the H+-binding sites          32
to either side of the membrane during turnover. Furthermore,
sugar binding or generation of a H+ electrochemical gradient            Structure and Catalytic Mechanism of the E. coli Maltose
(interior negative) increases the probability of opening on the         Transporter
periplasmic side of the membrane.                                       Amy Davidson, Cedric Orelle, Jinming Cui, Frances
30                                                                      Chemistry, Purdue University, 560 Oval Drive, West
                                                                        Lafayette, IN, 47907
Exploring integral membrane protein structure and                       The maltose/maltodextrin transport system from E. coli has
folding                                                                 proven to be an excellent model system for study of the
Charles Brooks                                                          structure and mechanism of ATP-binding cassette transporters.
Chemistry and Biophysics, University of Michigan, 930 N.                In our lab, we have used site-directed spin labeling and
University Ave, Ann Arbor, MI, 48109                                    electron paramagnetic resonance spectroscopy to better
In this talk I will discuss the development of an implicit              understand the mechanism of maltose uptake. Nitroxide
solvent/implicit membrane model and a range of applications             spin labels are attached via cysteine residues substituted at
of this model to explore the insertion, folding and assembly            one or more positions in the transporter and conformational
of membrane proteins. The applications will cover a range of            changes induced by the addition of substrates or effectors to
systems from single trans-membrane helical domains to the               the transporter. Changes in distance between two nitroxides
assembly and modeling of GPCRs.                                         attached to a single transporter and changes in mobility at the
                                                                        site of spin labeling are interpreted in light of several high
31                                                                      resolution structures of the maltose transporter.

The molecular function and dysfunction of membrane                      33
attack complex / perforin-like (MACPF) proteins
James Whisstock                                                         The structural basis for P-TEFb (Cdk9/cyclin T)
Department of Biochemistry and Molecular Biology and ARC                recognition properties
Centre of Excellence in Structural and Functional Microbial             Louise Johnson1,2, Sonja Baumli1
Genomics, Monash University, Clayton, Australia                         1
                                                                         Molecular Biophysics, University of Oxford, Biochemistry
The Membrane Attack Complex / Perforin-like (MACPF)                     Department, Parks Road, Oxford, OX1 3QU, United
proteins perforin and complement C9 are mammalian pore                  Kingdom, 2Diamond Light Source, Harwell Science and
forming molecules associated with the immune response                   Innovation Campus, Didcot, OX11 0DE, United Kingdom
against viruses and pathogenic bacteria respectively. Further,          Fred Richards was a marvelous, charismatic scientist, a
recent studies suggest that perforin may be important for               leader in the field of protein science who made outstanding
tumour surveillance and in eliminating pre-cancerous cells.             contributions to our understanding of protein folding and
Recently, we showed that MACPF proteins are distantly                   organisation. The postdoctoral year 1966 that I spent I his
homologous to cholesterol dependent cytolysins (CDCs), an               laboratory was unforgettable. Later he came to Oxford to
important family of bacterial pore forming toxins. Based upon           spend a Sabbatical year with David Phillips, a year full of
these data it is suggested that, like CDCs, MACPF proteins              fun that led to the construction of the Richards Box. Protein
may firstly oligomerise on the surface of the membrane to               kinases catalyze key phosphorylation reactions in signalling
form a pre-pore form. Followed pre-pore formation it is                 cascades that affect nearly every aspect of the cell’s life. The
suggested that a concerted conformational change permits                lecture will describe our work on the cyclin dependent protein
two clusters of α-helices to unwind and insert into the                 kinases (CDKs) and the kinase involved in the regulation of
membrane as amphipathic β-strands. Here, using structural,              transcription, CDK9/cyclin T (P-TEFb Positive-Transcription
biophysical and mutagenesis studies we test this hypothesis             Elongation Factor b). Protein kinases have become prime
and investigate how perforin as well as related MACPF-                  targets for drug intervention in cancer and the lecture will
domain-containing proteins form membrane inserted pores.                review some of the structural aspects of protein kinase
We also investigate how certain mutations in MACPF                      inhibitors that have been approved for clinical use.
proteins result in dysfunction and disease. In particular, it is
suggested that many perforin mutations may interfere with
proper perforin folding whereas other mutations may directly
interfere with the mechanism of membrane insertion.


34                                                                      singly or in combination, with key hydrophobic residues on
                                                                        the target CaMBDs. The recently determined novel structure
Fred Richards, Structural Biology, and the Ribosome                     of the 77-residue pre-IQ–IQ regulatory segment with bound
Peter Moore                                                             Ca2+·CaM best exemplifies CaM’s versatility; four Ca2+·CaM
Chemistry, Yale University, 255 Prospect St., New Haven,                molecules are bound in two different sets of conformations,
CT, 06520-8107                                                          one set bridging a dimer of two coiled-coil pre-IQ domains
Even though Perutz and Kendrew demonstrated the awesome                 and the other enfolding the IQ domain.
power of macromolecular crystallography in the 1960s,
in the 1970s most biochemists still thought it a costly,                36
exotic technique, and study sections were reluctant to fund
it. About 1975, Fred Richards, who happens to have been                 Transcription factor-DNA interactions: cis regulatory
as fine a scientific administrator as he was a scientist, put           codes in genomes
crystallography on a sound footing at Yale by organizing                Martha Bulyk
a consortium of faculty that under his leadership worked                Medicine/Genetics, Brigham and Women’s Hospital/Harvard
together on crystallographic and scattering problems for the            Medical School, NRB 466D, 77 Avenue Louis Pasteur,
next quarter of a century. As evidence of Fred’s success in this        Boston, MA, 02115
endeavor one might cite the structure of the large ribosomal            The interactions between transcription factors (TFs) and
subunit, which was published in 2000, at about the time Fred            their DNA binding sites are an integral part of the regulatory
retired from science. In addition to commenting on this facet           networks within cells. Identification of the DNA binding
of Fred’s career, I will discuss some recent findings about the         specificities of sequence-specific TFs is important for
way antibiotics inhibit the protein synthetic activity of the           understanding transcriptional regulatory networks, in
ribosome.                                                               particular for the prediction of cis regulatory modules (e.g.,
                                                                        transcriptional enhancers), inference of cis regulatory codes,
35                                                                      and interpretation of in vivo TF occupancy data and gene
                                                                        expression data. Using highly parallel in vitro technology
Calmodulin: Bound Structures                                            developed in my group termed universal protein binding
Florante Quiocho, Jennifer Fallon                                       microarrays (PBMs), we have characterized the sequence
Biochemistry & Molecular Biology, Baylor College of                     specificities of DNA-protein interactions at high resolution
Medicine, One Baylor Plaza, Houston, TX, 77030                          for >500 TFs from a wide range of species. These data have
Calmodulin (CaM) (17 kDa) is the most versatile Ca2+ sensor             permitted us to identify novel TFs and their functions, to
and regulatory protein, targeting over 100 enzymes and                  investigate how TFs interact with their target sites in vivo, and
effector proteins associated with many diverse biological               to identify features of transcriptional enhancers important for
and cellular functions. The versatility of CaM is derived               driving appropriate gene expression patterns in metazoans.
from its biochemical and structural features. The structural            We anticipate that further analyses of closely related TFs and
features will be the topic of the talk. In 1985, the initial            the cis regulatory elements in which their binding sites occur
crystal structure of the Ca2+-loaded CaM (or Ca2+·CaM)                  will reveal additional features of cis regulatory codes and
showed an elongated dumbbell-shaped molecule in which                   how TFs within gene families diverge to acquire new DNA
the N- and C-terminal lobes, each with two EF-hand Ca2+-                binding specificities and functions.
binding sites, were separated by a solvent-exposed linker
α-helix. Beginning in 1992, our laboratory has determined               37
several crystal structures of native Ca2+·CaM bound to the
CaM-binding domains (CaMBDs) of two kinases, the                        A massively-parallel microchemostat platform for single
neuronal subunit a1 of the V0 ATPase, and the IQ and the                cell imaging
combined pre-IQ–IQ regulatory segments at the C-terminus                Sebastian Maerkl
of the cardiac L-type (Cav1.2) calcium channel. We have also            Institute of Bioengineering, Ecole Polytechnique Federale
determined the structures of active Ca2+·CaM linker mutants,            de Lausanne, EPFL-STI-IBI-LBNC, Station 17, BM 2111,
with the linker helix shortened by two or three residues or             Lausanne, 1015, Switzerland
replaced by the longer troponin C linker helix, bound to a              We have developed a microfluidic platform consisting of
kinase CaMBD. In all these bound structures, the Ca2+·CaM is            1056 microchemostats. Each chemostat is roughly 200μm x
no longer elongated, but transformed into collapsed or more             200μm in size and is continuously perfused with media. We
compact states with the two lobes differing in proximity and            actively program the device with cells ranging from bacteria
orientation (parallel or anti-parallel) depending on the nature         to yeast using an arraying method, enabling us to interrogate
of the targets. The relative positioning of the two lobes,              over 1000 different strains or clones in parallel on a single
which is achieved by variable uncoiling of the linker helix,            device. In our first proof of concept screen we have made use
enable the hydrophobic pockets in the two lobes to interact,            of the GFP-tagged S. cerevisiae strain library to allow us to


follow the expression and localization dynamics of over 4500           these networks is affected by external forces, such as those
proteins in yeast. We gathered steady state dynamics under             transmitted through the extracellular matrix or encountered
standard growth conditions, and perturbed the system by the            during cell movements, is not well understood. To investigate
addition of MMS to induce DNA damage and its cognate                   how mechanical boundary conditions affect cytoskeletal
cellular response pathways. With our massively-parallel                assembly and function, we have used atomic force microscopy
microchemostat platform it is now possible to follow the               to study branched actin networks reconstituted in vitro. The
expression and localization of thousands of proteins in vivo           branched networks are nucleated and confined between the
and on the single cell level with high-temporal and spatial            cantilever and a surface so that their growth rate and force
resolution, paving the way towards global analysis of the              can be quantified by cantilever deflection. Our results
dynamics of protein regulation.                                        suggest that mechanical force and displacement play a crucial
                                                                       role in organizing branched actin networks and defining
38                                                                     their properties, raising questions about how the history
                                                                       of mechanical interactions of cells with their environment
Electro-optical trapping of single molecules at the 1                  affects their cytoskeleton and behavior.
nanometer limit
Adam Cohen, Alexander Fields                                           40
Chemistry and Chemical Biology, Harvard University, 12
Oxford Street, Cambridge, MA, 02138                                    Structure and mechanism of bacterial transition metal
The anti-Brownian electrokinetic (ABEL) trap uses real-time            sensing transcriptional regulators
particle tracking and electrokinetic feedback to suppress              David Giedroc
the Brownian motion of a single molecule in free solution.             Chemistry, Indiana University, 800 E. Kirkwood Avenue,
The spectroscopy and dynamics of the molecule can then be              Bloomington, IN, 47405-7102
observed for an extended time without the perils of surface            A panel of metalloregulatory or “metal sensor” proteins
immobilization. Previous incarnations of the ABEL trap                 collectively coordinates the expression of genes that allow
have been used to study polymer mechanics and protein                  organisms to quickly adapt to chronic toxicity or deprivation
conformational dynamics, but were only able to trap objects            of both biologically essential transition metal ions and
with hydrodynamic diameters greater than 15 nm. The median             heavy metal pollutants found in their microenvironment.
size of human proteins is 375 amino acids, corresponding to            Accumulating evidence suggests that metal ion homeostasis
a diameter of ~1.8 nm, and so most proteins could not be               and resistance define an important tug-of-war in human
trapped by any means. We now demonstrate stable trapping               host-bacterial pathogen interactions. This adaptive response
in aqueous buffer of the 1.3 kDa dye Alexa Fluor 647, one of           originates with the formation of specific metal coordination
the smallest objects one could hope to trap and a greater than         complexes that drives protein quaternary structural switching
600-fold reduction in mass relative to past accomplishments.           within a metal sensor oligomer. Recent insights into our
This achievement opens the possibility to trap and study any           understanding of metal sensor proteins as specialized, metal-
fluorescently labeled molecule in free solution.                       selective allosteric proteins will be discussed, as will efforts
We present a novel statistical framework to calculate transport        to understand the structural and physical basis of how metal
properties of trapped molecules, and demonstrate its ability to        binding drives allosteric inhibition or activation of operator
distinguish a mixture of two species with differing diffusion          DNA binding using NMR spectroscopy, thermodynamic
coefficient. Together, these advances greatly extend the size          methods and native chemical ligation strategies. These
range of molecules that can be studied by room temperature             studies of a paradigm ArsR family Zn(II) efflux repressor
feedback trapping, and impart a new ability to measure the             from S. aureus, CzrA and a Cu(I)-specific sensor from
diffusion coefficient of each trapped molecule. We anticipate          Mycobacterium tuberculosis, CsoR, are consistent with the
that these tools can be applied to the study of single-molecule        hypothesis that the structure of the first coordination shell
conformational and binding dynamics.                                   dictates metal selectivity, which in turn establishes a “second”
                                                                       coordination shell that mediates physical linkage of the metal
39                                                                     and DNA binding sites. Supported by a grant from the NIH
                                                                       (R01 GM042569).
Probing the mechanical response of actin networks with
atomic force microscopy
Daniel Fletcher
Bioengineering, UC Berkeley, QB3 Institute, 608 Stanley
Hall #3220, Berkeley, CA, 94720
Organized networks of actin filaments drive cellular
protrusions and intracellular movements, as well as contribute
to the mechanical rigidity of cells. How the architecture of


41                                                                     43
Single-Molecule Dynamics of Metal Regulators and                       How Aliphatic Halogenases Prevent Competing
Transporters                                                           Hydroxylation
Peng Chen                                                              J. Bollinger, Jr. 1, Megan Matthews2, Linde Miles2, Carsten
Chemistry and Chemical Biology, Cornell University, Baker              Krebs3, Tyler Grove4, Squire Booker5, Christopher Neumann6,
Lab, Ithaca, NY, 14853                                                 Christopher Walsh6
To maintain normal metal metabolism, organisms utilize
                                                                         Chemistry and Biochemistry and Molecular Biology, Penn
dynamic cooperation of many biomacromolecules for                      State University, 336 Chemistry Building, University Park,
regulating metal ion concentrations and bioavailability.               PA, 16802, 2Chemistry, Penn State University, 318 Chemistry
How these biomacromolecules work together to achieve                   Building, University Park, PA, 16801, 3Chemistry and
their functions is largely unclear, however. For example,              Biochemistry and Molecular Biology, Penn State University,
how do metalloregulators and DNA interact to control gene              332 Chemistry Building, University Park, PA, 16802,
expression to maintain healthy cellular metal level? How
                                                                         Chemistry, Penn State University, 327 Chemistry Building,
do metal chaperones and transporters collaborate to deliver            University Park, PA, 16801, 5Chemistry and Biochemistry
metal ions?                                                            and Molecular Biology, Penn State University, 302 Chemistry
                                                                       Building, University Park, PA, 16802, 6Biological Chemistry
Our group has been studying the macromolecular machineries             and Molecular Pharmacology, Harvard Medical School, 240
for metal regulation and transport at the singe-molecule level.        Longwood Ave, Boston, MA, 02115
For metal regulation, we have developed engineered DNA
Holliday junctions as general single-molecule reporters for            The Fe(II)- and α-ketoglutarate-dependent hydroxylases and
studying metalloregulator-DNA interactions. We show how                halogenases employ similar reaction mechanisms involving
MerR-family regulators, in particular the Pb2+-responsive              hydrogen-abstracting Fe(IV)-oxo intermediates.1 A crucial
regulator PbrR691 and the Cu1+-responsive regulator                    distinction between their active sites is the replacement of
CueR, bind to DNA, changes DNA structure, constrains                   the carboxylate residue from the His2(Asp/Glu)1 “facial
DNA conformational flexibility, and alters DNA dynamics                triad” of iron ligands found in the hydroxylases by alanine
for transcriptional regulation. For metal transport, we have           in the halogenases, which vacates a site for a halide ion
developed nanovesicle trapping coupled with single-molecule            (Cl– or Br–) to coordinate.2 The Scheme illustrates how
fluorescence-resonance-energy-transfer (smFRET) to study               the divergent outcomes have been rationalized to result
the weak, transient protein interactions between the copper            from alternative “radical-rebound” steps: hydroxyl radical
chaperone Hah1 and the transporter Wilson disease protein.             in the hydroxylases (top) versus halogen (X) radical in
We show how they interact dynamically forming multiple                 the halogenases (bottom).2,3 The basis for strict rebound
transient intermediates and how their interactions are coupled         specificity of the halogenases is not known but could reflect,
to copper transfer.                                                    at least in part, an inherently greater rebound reactivity of
                                                                       the coordinated halogen. According to this notion and the
42                                                                     Scheme, one might anticipate hydroxylation in a halogenase
                                                                       variant with the carboxylate residue restored by mutagenesis.
Crystallographic snapshots of metalloenzyme complexes                  To the contrary, the A118D/E variants of the halogenase
involved in biological carbon dioxide sequestration                    SyrB2 reportedly do not mediate hydroxylation.2 I will present
Cathy Drennan1, Yan Kung2, Nozomi Ando2, Tzanko                        an experimental analysis of the reactions of wild-type SyrB2
Doukov3, Gunes Bender4, Stephen Ragsdale4                              and its carboxylate-restored variant with different substrates,
  Chemistry and Biology, MIT/HHMI, 77 Massachusetts                    revealing the crucial role of substrate positioning in ensuring
Ave, 68-680, Cambridge, MA, 02139, 2Chemistry, MIT,                    selectivity for halogenation over hydroxylation.4
77 Massachusetts Ave, Cambridge, MA, 02139, 3SLAC,                     1. Krebs C, Galonić Fujimori D, Walsh CT, & Bollinger JM,
2575 Sand Hill Road, Menlo Park, CA, 94025, 4Biological                   Jr. (2007) Acc. Chem. Res. 40, 484-492.
Chemistry, University of Michigan, 1150 W. Medical Center
Dr., Ann Arbor, MI, 48109                                              2. Blasiak LC, Vaillancourt FH, Walsh CT, & Drennan CL
                                                                          (2006) Nature 440, 368-371.
Acetogenic bacteria play an important role in the global carbon
cycle by removing carbon dioxide from our environment and              3. Hanauske-Abel HM & Günzler V (1982) J. Theor. Biol.
using it as their carbon and energy source. In this work, we              94, 421-455.
investigate the structures of the key metalloenzymes involved          4. Matthews ML, Neumann CS, Miles LA, Grove TL, Booker
in acetogenesis, both to gain mechanistic insight as well as              SJ, Krebs C, Walsh CT & Bollinger JM Jr. (2009) Proc.
to understand the molecular movements that permit these                   Natl. Acad. Sci. U.S.A. 106, 17723-17728.
enzymes to act in concert. In particular, we will consider how
one-carbon units are passed from enzyme to enzyme to allow
for life on carbon dioxide.


44                                                                      study the in vivo maturation that led to m836, as well as the
                                                                        engineering process that generated our potent optimized HFA
The evolution of new protein folds by straightforward                   anti-human IL-13 antibody.
mutational mechanisms
Matthew Cordes                                                          46
Chemistry and Biochemistry, University of Arizona, BSW
439, 1041 E. Lowell St., Tucson, AZ, 857210088                          Structure-guided SCHEMA recombination generates
There are over a thousand known protein folds. While many               highly stable and functionally diverse enzyme families
different folds may have arisen by independent pathways and             Frances Arnold
processes, some structures have evolved from others through             Chemical Engineering MC 210-41, California Institute of
topological rearrangements induced by mutations. I will                 Technology, 1200 E. California Blvd., Pasadena, CA, 91125
discuss two cases of fold evolution in which bioinformatic              We are investigating ways to emulate evolution in the
approaches have allowed targeting, structural characterization          laboratory in order to create new proteins with desirable
and comparison of pairs of homologs with significant                    properties. This approach circumvents our profound
sequence similarity but different folds. One case involves              ignorance of how the amino acid sequence encodes protein
replacement of α-helical secondary structure with β-sheet in            function and exploits the ability of biological systems to
the Cro protein family; the other involves the swapping and             evolve and adapt. Here I will show how structure-guided
register shifting of β-strands within the β-barrel structures           SCHEMA recombination generates novel chimeric enzymes
of insect salivary lipocalins. In both instances, sequence              having dozens of mutations relative to their closest parent
and structure comparisons support a homologous switching                sequences. SCHEMA identifies the crossovers that break
mechanism, in which a significant region of the sequence                the fewest interactions, and as a result SCHEMA blocks
has undergone major structural rearrangement in response                contribute in a simple linear fashion to overall protein
to accumulation of amino-acid substitutions and/or small                stability. The most stable recombinants can therefore be
insertion and deletion events.                                          predicted with high reliability by sampling a small fraction of
                                                                        the chimeric protein library. This approach has been used to
45                                                                      generate dozens of new, highly stable enzymes from different
                                                                        structural and functional classes, including cellulases (three
In Vivo and In Vitro Evolution of an Anti-human IL-13                   different families) and cytochrome P450s. We have used
Antibody                                                                these synthetic protein families to rapidly pinpoint key
Johan Fransson                                                          stabilizing residues and interactions. The resulting stable
Biologics Research, Centocor R&D, Johnson and Johnson,                  enzyme chimeras also exhibit (potentially useful) functional
3210 Merryfield Row, San Diego, CA, 92121                               diversity.
Therapeutic antibodies are rapidly expanding in the portfolios
of pharmaceutical companies. To be competitive in this                  47
field, the need for optimized and highly potent antibodies is
critical and evolving antibodies beyond their natural affinities        Structural Studies of the Amyloid State
is becoming commonplace in therapeutic programs. We                     David Eisenberg, Michael Sawaya, Arthur Laganowsky,
identified an antibody with high affinity (50 pM) to human IL-          James Stroud, Lukasz Goldschmidt, Meytal Landau, Poh
13 generated by mouse immunization. This antibody, denoted              Teng, Marcin Aposotol, Angie Soriaga
m836, was humanized by a proprietary methodology called                 UCLA-DOE Institute for Genomics and Proteomics, Box
Human Framework Adaptation (HFA), which consists of                     951570, Los Angeles, CA, 90095-1570
Human Framework Selection (HFS) followed by Specificity-                Structural studies of amyloid fibrils and small oligomers
Determining Residue Optimization (SDRO). During HFS,                    will be described. Some 60 atomic structures have been
intact human germline framework regions were combined                   determined of fibril-forming segments of amyloid-forming
with mouse residues located in or near the antigen-binding              proteins. These segments come from 10 fibril-forming
site to create a small library of HFS variants. After screening         proteins, each associated with an amyloid disease. The
the HFS library, we identified several variants with affinities         common characteristic of these structures is a pair of tightly
in the 250 pM range. To restore the affinity back to that of the        interdigitating beta sheets, with the sidechains of one sheet
mouse antibody, combinatorial Fab libraries were designed               intermeshing with those of the mating sheet. The interfaces
with diversity in SDRs and cloned into a Fab-pIX vector for             are without water. A given sequence segment can form more
phage display. The SDRO step resulted in the enrichment of              than one structure, suggesting the molecular basis of amyloid
variants with affinities in the single-digit picomolar range.           polymorphism and prion strains. These structures, coupled
Crystal structures of Fabs in complex with IL-13 were                   to biochemical and EM studies, have permitted us to identify
obtained for m836, the HFS variant chosen for SDRO, and                 the features of proteins that cause them to form fibrils.
one of the highest affinity SDRO variants. This enabled us to


References:                                                             49
Goldschmidt L, Teng PK, Riek R, Eisenberg D. (2010).
Identifying the amylome, proteins capable of forming                    Determination of the Structure of an Amyloid Protofibril
amyloid-like fibrils. Proc. Natl. Acad. Sci. U.S.A.. Feb 2010.          with Magic Angle Spinning NMR
107(8):3487-92.                                                         Robert Griffin
                                                                        Francis Bitter Magnet Lab and Chemistry, Massachusetts
Teng PK, Eisenberg D. (2009). Short protein segments can                Institute of Technology, Room NW14-3220, MIT, 77
drive a non-fibrillizing protein into the amyloid state. Protein        Massachusetts Avenue, Cambridge, MA, 02139
Eng. Des. Sel.. Aug 2009. 22(8):531-6.
                                                                        Many biological systems form nanostructures that are well
Wiltzius JJ, Landau M, Nelson R, Sawaya MR, Apostol                     ordered on a microscopic but not a macroscopic scale, and are
MI, Goldschmidt L, Soriaga AB, Cascio D, Rajashankar K,                 therefore difficult to examine with standard tools of structural
Eisenberg D. (2009). Molecular mechanisms for protein-                  biology. A class of systems exhibiting these properties are
encoded inheritance. Nat. Struct. Mol. Biol.. Sep 2009.                 amyloid peptides and proteins that assemble into fibrils. In
16(9):973-8.                                                            this presentation we review approaches to determining the
Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers                 structure of these systems with high resolution magic angle
SA, Apostol MI, Thompson MJ, Balbirnie M, Wiltzius JJ,                  spinning (MAS) NMR techniques. Using these approaches
McFarlane HT, Madsen AØ, Riekel C, Eisenberg D. (2007).                 we determined the high resolution structure of two peptides
Atomic structures of amyloid cross-beta spines reveal varied            derived from transthyretin from a set of ~70 torsion angle
steric zippers. Nature. May 2007. 447(7143):453-7                       and distance constraints in each case. The spectra also
                                                                        permit determination of the arrangement of the monomers
48                                                                      that form sheets from a set of ~7 interstrand distances, and
                                                                        the sheets that assemble to form amyloid protofibrils from
Folding and aggregation of ALS-associated mutant Cu,Zn                  a series of ~12 intersheet distances. We combine the high
superoxide dismutases                                                   resolution molecular structure data with cryoEM and STEM
Elizabeth Meiering                                                      experiments to elucidate the structure of the fibril. In a second
Departments of Chemistry and Biology, Guelph-Waterloo                   set of experiments we discuss extensions of these MAS
Centre for Graduate Studies in Chemistry and Biochemistry,              techniques to structural studies of amyloidiogenic proteins,
University of Waterloo, 200 University Avenue West,                     in particular phophatidylinositol-3-kinase SH3 domain (PI3-
Waterloo, N2L 3G1, Canada                                               SH3) and β-2-microglobulin
Over 140 mutations in Cu,Zn superoxide dismutase (SOD1)
are known to cause familial amyotrophic lateral sclerosis               50
(fALS), a devastating and invariably fatal neurodegenerative
disease. SOD1 is a homodimeric Greek-key β-barrel                       Membrane binding and aggregation of alpha-synuclein in
protein, with each subunit containing one intramolecular                Parkinson’s disease
disulfide bond and binding one Cu and one Zn ion. Thus,                 Jean-Christophe Rochet1, Katherine Strathearn1, Amy
SOD1 can exist in multiple forms in vivo that differ with               Griggs1, Jagadish Hindupur1, Jeremy Schieler1, Katie Ruf1,
respect to metallation, disulfide bond formation, and subunit           Anjan Pandey2, Farzin Haque2, Jennifer Hovis2, Joseph
association. A prominent disease hypothesis is that SOD1                Irudayaraj3
mutants cause ALS by promoting toxic aberrant protein
                                                                         Medicinal Chemistry and Molecular Pharmacology, Purdue
aggregation, analogous to many other protein misfolding                 University, 575 Stadium Mall Drive, RHPH 410A, West
diseases including Alzheimer’s, Parkinson’s, Huntington’s               Lafayette, IN, 47907, 2Chemistry, Purdue University, 560
and prion diseases. We have undertaken a systematic                     Oval Drive, West Lafayette, IN, 47907, 3Agricultural &
investigation of the thermodynamics and kinetics of the                 Biological Engineering, Purdue University, 225 S. University
folding and aggregation of different forms of SOD1.                     Street, West Lafayette, IN, 47907
Extensive data reveal that different mutations have complex             Parkinson’s disease (PD) results from a progressive loss
and highly variable effects on different forms of the protein.          of dopaminergic neurons from the substantia nigra in the
These effects can be rationalized in terms of a model whereby           midbrain. The postmortem brains of Parkinson’s patients
multiple pathways involving different forms of SOD1 can                 are characterized by oxidative damage and the presence of
lead to increased aggregation. I will discuss how variations            inclusions named Lewy bodies. Lewy body inclusions consist
in protein biophysical properties and pathways may be related           largely of amyloid-like fibrils formed by alpha-synuclein, a
to the distinct disease characteristics that are associated with        presynaptic protein with an affinity for membranes. Mutations
different SOD1 mutations.                                               in the gene encoding alpha-synuclein are associated with
                                                                        early-onset, familial PD. The disease-linked mutants have
                                                                        a greater tendency to form aggregates than the wild-type
                                                                        protein, suggesting that alpha-synuclein self-assembly plays


a role in neurodegeneration. We have shown that alpha-                  Mu, T-W.; Ong, D.S.T.; Wang, Y-J; Balch, W. E.; Yates,
synuclein undergoes post-translational modifications and                J.R.; Segatori, L.; Kelly, J.W. .”Chemical and Biological
forms membrane-bound aggregates in neuronal cells subjected             Approaches Synergize to Ameliorate Protein-Folding
to oxidative stress. When added to a supported lipid bilayer,           Diseases” Cell 2008 134, 769-781.
alpha-synuclein induces demixing of anionic and zwitterionic            Mu, T-W.; Fowler, D. M.; Kelly, J. W. “Partial Restoration
phospholipids, and it forms clusters that are closely associated        of Mutant Enzyme Homeostasis in Three Distinct Lysosomal
with regions enriched with anionic lipids. Alpha-synuclein              Storage Disease Cell Lines by Altering Calcium Homeostasis”
also undergoes accelerated self-assembly in the presence of             PloS Biol. 2008 6, 253-265, e26
synthetic phospholipid vesicles, resulting in the formation of
membrane-bound oligomers. From these observations, we                   Wiseman, R.L.; Powers, E.T.; Buxbaum, J.N.; Kelly, J.W.;
infer that phospholipid membranes stimulate the formation               Balch, W.E. “An Adaptable Standard For Protein Export
of alpha-synuclein aggregates. Current efforts are focused              from the Endoplasmic Reticulum” Cell 2007 131, 809-821.
on monitoring site-specific conformational changes that                 Cohen, E.; Cohen, T-B; Paulsson, J.F.; Du, D.; Estepa, G.;
accompany alpha-synuclein self-assembly at the membrane                 Pham, H.M.; Kelly, J.W.; Masliah, E.; Dillin, A. “Reduced
surface using the environment-sensitive fluorophore pyrene.             IGF-1 Signaling Delays Alzheimer’s-like Disease Progression
We are also using site-directed mutagenesis to investigate              in Mice” Cell 2009 137, 1157-1169.
how membrane binding and post-translational modifications               Powers, E.T.; Morimoto, R.I.; Dillin, A.; Kelly, J.W.; Balch,
impact the formation of neurotoxic alpha-synuclein                      W.E. “Biological and Chemical Approaches to Diseases
assemblies in cellular models of PD. These findings provide             of Proteostasis Deficiency” Ann. Rev. Biochem. 2009 78,
insight into the role of alpha-synuclein aggregation in PD,             959-91.
and they suggest strategies for discovering new therapies.
                                                                        Expanding the Synthetic Capabilities of the Cell
Adapting Proteostasis to Ameliorate Loss- and Gain-of-                  Virginia Cornish
Function Diseases                                                       Department of Chemistry, Columbia University, 3000
Jeffrey Kelly                                                           Broadway, New York, NY, 10027
Depts. of Chemistry and Molecular and Experimental
                                                                        The process of directed evolution successfully harnesses
Medicine, and The Skaggs Institute of Chemical Biology,
                                                                        recombinant DNA technology to engineer biomolecules
The Scripps Research Institute, 10550 North Torrey Pines
                                                                        with new functions in vitro through repeated cycles of
Rd., La Jolla, CA, 92037
                                                                        combinatorial DNA mutagenesis and high-throughput assay.
The chemical information encoded by the polypeptide                     To provide a breakthrough for the emerging demands of
sequence, co-translational modifications including N-linked             synthetic biology, my laboratory is engineering yeast so that
glycosylation, as well as interactions of the polypeptide               both the mutagenesis and selection steps of directed evolution
with proteostasis network components determine whether                  can be carried out entirely in vivo, under conditions of sexual
a protein will fold and function, be degraded, remain                   reproduction. Here I will describe chemical complementation,
intrinsically disordered or aggregate and create additional             which allows chemistry beyond that natural to the cell to be
proteostatic challenges for the organism. The proteostasis              linked to powerful growth selections using the yeast n-hybrid
network, comprising transcriptional and translational control           assay. Furthermore, I will present the design of a heritable
of protein synthesis, chaperone- and enzyme-assisted                    recombination system that enables genetic interchange of
folding, trafficking, disaggregation activities and degradation         beneficial mutations among evolving populations.
activities will be introduced. The outset of the seminar will
focus on how the proteostasis network can be adapted to                 53
enable misfolding-prone proteins to fold, traffic and function
to ameliorate loss-of-function diseases such as the lysosomal           Structural and Dynamic Basis for the Assembly of Protein
storage disorders. The second half of the talk will focus on            Machineries by NMR
how proteostasis capacity can be enhanced to prevent gain-              Charalampos Kalodimos
of-function diseases associated with protein aggregation                Chemistry & Chemical Biology, Rutgers University, 599
and tissue degeneration, including Alzheimer’s and related              Taylor Rd, Piscataway, NJ, 08854
amyloid diseases.                                                       We will discuss the application of NMR spectroscopy to
References:                                                             characterizing supramolecular protein systems. Our lab
Balch, W.E.; Morimoto, R.I.; Dillin, A.; Kelly, J.W. “Adapting          is actively applying NMR to determine the functional
Proteostasis For Disease Intervention” Science 2008 319,                mechanisms of many machineries: the protein translocase, the
916-919                                                                 transcription machinery and the type III secretion machinery.
                                                                        We will primarily discuss the recognition and transport


mechanisms in the Sec translocase that involves the assembly            Dumpty. This talk will recap a career path that has led from
of chaperones, ATPase motors, and a membraneous channel.                a wholly reductionist approach to sequence information,
                                                                        through several intermediates, and ultimately to the current
54                                                                      research in our lab, which tackles protein folding in cellular
                                                                        environments. Key factors that are emerging as paramount
Excited states and (NMR) relaxation                                                                          in cellular folding are
Peter Wright                                                                                                 the ubiquitous weak
Molecular Biology, The Scripps Research Institute, 10550 N.                                                  intermolecular
Torrey Pines Road, MB-2, La Jolla, CA, 92037                                                                 associations and the
Molecular motions are central to the biological functions                                                    networks of chaperones
of proteins. Detailed knowledge of the dynamics and                                                          that     remodel      the
thermodynamics of proteins and of the kinetics of                                                            folding       landscape
conformational fluctuations is essential if we are to understand                                             and impact fluxes of
the relationships between protein structure and function.                                                    protein to any possible
NMR relaxation experiments provide a detailed description                                                    cellular fates: proper
of protein dynamics on a broad range of time scales, ranging                                                 folding, degradation, or
from ps to ms and beyond, and provide direct insights into the                                               aggregation.
protein energy landscape. In particular, relaxation dispersion
experiments permit quantitative analysis of the dynamics and            56
thermodynamics of slow conformational fluctuations and of
the kinetics and mechanism of protein-ligand interactions               Grabbing the cat by the tail: Discrete steps by a DNA
and protein folding events. Applications to the enzyme                  packaging motor and the inter-subunit coordination in a
dihydrofolate reductase (DHFR) show that progress through               ring-ATPase
the catalytic cycle involves a dynamic energy landscape,                C. Bustamante, J. Moffitt
where each intermediate populates excited states in which               University of California, Berkeley, CA, 94704
the protein conformation corresponds to that of the preceding           As part of their infection cycle, many viruses must package
or following intermediate in the cycle. Conformational                  their newly replicated genomes inside a protein capsid.
fluctuations in the active site loops, on a time scale that             Bacteriophage phi29 packages its 6.6 mm long double-
is directly relevant to catalysis, appear to be harnessed by            stranded DNA into a 42 nm dia. x 54 nm high capsid using a
the enzyme to control the flux of substrate, product, and               multimeric ring motor that belongs to the ASCE (Additional
cofactor. Relaxation dispersion also provides novel insights            Strand, Conserved E) superfamily of ATPases. A number of
into mechanisms of protein folding and unfolding and into               fundamental questions remain as to the coordination of the
the coupled folding and binding processes that mediate the              various subunits in these multimeric rings. The portal motor
physiological functions of many intrinsically disordered                in bacteriophage phi29 is ideal to investigate these questions
proteins.                                                               and is a remarkable machine that must overcome entropic,
                                                                        electrostatic, and DNA bending energies to package its
55                                                                      genome to near-crystalline density inside the capsid. Using
                                                                        optical tweezers, we find that this motor can work against
From reductionist to post-reductionist: a protein folding               loads of up to ~55 picoNewtons on average, making it one of
pathway                                                                 the strongest molecular motors ever reported. We establish
Lila Gierasch                                                           the force-velocity relationship of the motor. Interestingly,
Biochem & Mol Biology and Chemistry, Univ of                            the packaging rate decreases as the prohead fills, indicating
Massachusetts Amherst, 710 N Pleasant St, Amherst, MA,                  that an internal pressure builds up due to DNA compression
01003                                                                   attaining the value of ~6 MegaPascals at the end of the
The complexity of the cellular environment is a fact of life.           packaging. We show that the chemical energy of ATP is
Proteins are selected to fold and perform their functions under         converted into mechanical work during phosphate release.
the constraints of this environment, including macromolecular           Using ultra-high resolution optical tweezers, we determined
crowding, spatial organization, weak yet specific interactions,         the step size of the motor and established the coordination of
and more. Those of us seeking to decipher the information in            the ATPases around the ring. We propose a comprehensive
protein sequences must admit these physiological realities              model of the operation of this motor.
and adopt a ‘post-reductionist’ view of protein science.
Many technical and theoretical challenges accompany any
efforts to explore protein folding and function under in-vivo
conditions, as even gentle disruption of the cellular context
irreversibly abolishes its essential features, not unlike Humpty


57                                                                        58
Dynamics and Stability in Virus Maturation: Harnessing                    Structural Biology of Type IV Secretion Systems
a Molecular Machine                                                       Gabriel Waksman
John (Jack) Johnson                                                       ISMB, Malet street, London, WC1E 7HX, United Kingdom
Molecular Biology, The Scripps Research Institute, 10550 N.               Type IV secretion systems (T4SSs) are molecular machines
Torrey Pines Rd., La Jolla, CA, 92037                                     used for the transport of macromolecules across the bacterial
Assembly of quasi-equivalent virus capsids engages molecular              cell envelope in Gram-negative bacteria. T4SSs are highly
switches to create different interface contacts between the               versatile. Conjugative T4SSs translocate DNA from a donor
same gene products. The particle often assembles as a fragile,            to a recipient bacterium and contribute to bacterial genome
spherical shell in which the subunits are properly positioned             plasticity, spread of antibiotic resistance or other virulence
on the appropriate surface lattice and then quasi-equivalent              trait among bacterial pathogens. In some bacteria such as
subunit contacts differentiate during maturation, creating a              Helicobacter pylori (Cag PI), Brucella suis (VirB/D), or
robust, faceted particle. Folding of the switch regions of the            Legionella pneumophila (Dot, Icm), T4SSs are directly
subunit depends on assembly and maturation that are affected              involved in pathogenicity as they mediate the secretion
by biochemical cues. NωV is a quasi-equivalent virus,                of virulence factors (DNA or toxins) into host cells (1).
with a T=4 surface lattice, where this process is dramatic (a             The archetypal T4SS, the VirB/D system, was defined in
change in particle size of 100Å during maturation) and can be             Agrobacterium tumefaciens where it is naturally responsible
investigated in vitro. Here we use biochemistry, Small Angle              for the delivery of the T-DNA to the plant host-cell. The A.
X-ray Scattering and electron cryo-microscopy and image                   tumefasciens VirB/D system comprises 12 proteins (VirB1 to
reconstruction (CryoEM) to show that regions of NωV                  11 and VirD4) (1). In the past few years, atomic structures of
subunit folding are assembly-dependent and occur at rates                 isolated components such as VirD4, VirB11, VirB5, virB8,
determined by their quasi-equivalent position in the capsid.              virB10 and virB9 have become available and have provided
NωV matures when virus-like-particles (VLPs) isolated                insights into the mechanism of T4SS assembly and substrate
from an expression system at pH 7.6 are lowered to pH 5.0.                secretion (1). However, no structural data was available
Kinetics of maturation dependent, auto-catalytic cleavage                 concerning the assembly of the complex, particularly at the
are complex and suggest the four, quasi-equivalent, subunits              level of both bacterial membranes where the T4SS is supposed
cleave at different rates1. Difference cryoEM electron density            to form pores/channels. We here present the cryoEM structure
maps computed at three time points following maturation                   of a 1 MDa complex composed of VirB7, VirB9 and VirB10
initiation revealed that the active site for the cleavage                 homologues from the E. coli conjugative plasmid pKM101
was formed at different rates in the four subunits in the                 T4SS (2). We also present the crystal structure of a 0.6 MDa
icosahedral asymmetric unit, explaining the unusual kinetics              sub-complex representing the entire outer membrane T4SS
of the maturation cleavage2. Equilibrium Small Angle X-ray                channel (3). The molecular characterization of these ternary
Scattering (SAXS) experiments showed that many intermediate               complexes provide key insights into the type IV secretion
states could be populated with a homogeneous ensemble of                  systems architecture and assembly.
particles by carefully controlling the pH2. A titration curve for         1. Fronzes, R., Christie, P., and Waksman G. Nature Reviews
the LCC was generated that showed that the virtual pKa (i.e.                 Microbiology. 2009, 7:703
the composite of all titratable residues that contribute to the
LCC) of the particle is 5.8. Equilibrium SAXS measurements                2. Fronzes R., Schaefer E., Saibil H., Orlova E. and Waksman
also showed that the particle dimension between pH 5.5 and 5                 G., Science, 2009, 323, 266.
requires the autocatalytic cleavage to achieve its final compact          3. Chandran V., Fronzes, R., Duquerroy, S., Cronin, N.,
size. We suggest that a balance of electrostatic and structural              Navazza, J., and Waksman G. 2009, Nature. 462:1011-
forces shapes the energy landscape of the LCC with the latter                1015.
requiring “annealing” of portions of the subunit.
1. Matsui, T., Lander, G., and Johnson, J.E. 2009 Characterization
   of Large Conformational Changes and Auto-proteolysis in the
   Maturation of a T=4 Virus Capsid. J Virol 83, 1126-1134.
2. Matsui, T., Lander, G., Khayat, R. and Johnson, J.E. 2010
   Subunits fold at position-dependent rates during maturation
   of a eukaryotic RNA virus. submitted
3. Matsui, T., Tsuruta, H., & Johnson, J.E. 2010 Balanced
   Electrostatic and Structural Forces Guide the Large
   Conformational Change Associated with Maturation of a T=4
   Virus. Biophys J. in press


Amyloids and Protein Misfolding                                          expansion of CAG codon repeats, which code for polyQ in
                                                                         the corresponding gene products. In Huntington’s disease, the
(100 – 111)                                                              repeat-length threshold is about 37 glutamines. Our previous
                                                                         studies showed that aggregation rates of simple polyQ
100                                                                      peptides increase with repeat length, and that polyQ peptides
                                                                         with relatively long repeat lengths aggregate via a nucleated
Dynamic local unfolding in alpha-1 antitrypsin provides a                growth pathway with a critical nucleus (N*) of about one.
mechanism for serpin polymerization                                      We have hypothesized that this monomeric nucleus is a rare,
Beena Krishnan1, Lila Gierasch1,2                                        highly organized, folded structure resembling a small β-sheet
  Biochem. & Mol. Biol., 2Chemistry, UMass, Amherst, 710                 rich protein. We report here on the in vitro aggregation of a
N. Pleasant Street, Amherst, MA, 01003                                   short repeat length polyQ peptide, Q23, employing an HPLC-
Serine protease inhibitor (serpin) superfamily members                   sedimentation kinetics assay, and dynamic light scattering,
modulate proteolytic cascades in a wide array of physiological           EM, and FTIR measurements. We found for the Q23
systems. Key to serpin protease inhibitory function is a major           peptide that aggregation is very slow even at 100 – 200 μM
conformational transition triggered upon cleavage at a site              (concentrations much higher than required for longer polyQ
within the reactive centre loop (RCL). The released N-terminal           peptides), and that – as with other polyQ peptides – there
portion of the RCL, linked by acylation to the protease,                 are no non-amyloid aggregates formed at any time during
inserts into the central beta-sheet, thus enlarging it from five         the reaction. Reactions exhibit all the earmarks of nucleated
strands to six and converting the metastable native or ‘S’               growth reactions, including sensitivity to seeding and the
(stressed) state to a loop-inserted alternative fold complexed           existence of an equilibrium position and corresponding critical
with a disabled target protease. The conformational plasticity           concentration. Analysis of the concentration dependence of
of serpins underlies both their activities as protease inhibitors        the initial aggregation kinetics of the Q23 peptide surprisingly
and their susceptibility to pathogenic misfolding. Many                  revealed a multimeric nucleus consisting of four monomer
point mutations lead to accumulation of toxic misfolded                  peptide units. For comparison, we also repeated previously
serpin polymers, which are implicated in clinical conditions             reported kinetics studies on the aggregation of a longer
collectively termed serpinopathies. How the amino acid                   polyQ peptide, Q37, which confirmed the previous result of
sequences of serpins encode the metastable native S state and            an N* of about one. FTIR and EM studies confirm that the
the ability to undergo such a striking conformational switch             aggregates of both peptides are morphologically similar and
remains largely unknown. Here, we structurally characterize              rich in β-sheet. The results contribute to our understanding of
a sheet-opened state of the serpin alpha-1 antitrypsin (A1AT)            the underlying biophysics of repeat length effects in polyQ
populated in mildly destabilizing conditions to shed light               diseases, and may also hold clues to the structure of the
on features of the intermediate required for the structural              monomeric nucleus for pathological length polyglutamine
rearrangement of A1AT upon protease cleavage. In contrast                aggregation.
to previous proposals that the metastability of the S state
is due to uniformly distributed conformational lability,                 102
we identify a region that locally unfolds and allows A1AT
to be receptive to strand insertion. Mutations in A1AT that              Expression and Purification of Growth Factor Receptor
cause polymerization-induced serpinopathies map to this                  from Inclusion Bodies
labile region, suggesting that the evolution of a functional             Vivek Badwaik1, Jane Bartonjo2, Jesse Evans2, Chad Wills2,
mechanism required serpins to sample conformations on a                  Ajay Jain3, Shivendra Sahi3, Rajalingam Dakshinamurthy2
dynamic energy landscape and in so doing suffer increased
                                                                           Chemistry, WKU, 1555,Chestnut Street, APT # 36, Bowling
risk of aggregation.                                                     Green, KY, 42101, 2Chemistry, Western Kentucky University,
                                                                         Department of Chemistry, TCCW 420, 1906 College Heights
This work was supported by grants from the NIH (OD-00045
                                                                         Blvd. #11079, Bowling Green, KY, 42101, 3Biology, Western
to LMG) and the Alpha-1 Foundation (to BK).
                                                                         Kentucky University, Department of Biology, 1906 College
                                                                         Heights Blvd, Bowling Green, KY, 42101
                                                                         Fibroblast growth factors (FGFs) are a family of growth
Repeat-length dependent nucleus size and polyglutamine                   factors that are crucial in key cellular processes such as cell
aggregation kinetics                                                     proliferation, cell differentiation, wound healing, and tumor
Karunakar Kar, Ravindra Kodali, Ronald Wetzel                            growth. FGFs exert their biological activity by binding to
Structural Biology, PIND, University of Pittsburgh School of             their cell surface tyrosine kinase receptors (FGFRs). FGFRs
Medicine, 3501 Fifth Avenue, 2054 BST3, Pittsburgh, PA,                  contain an extracellular domain, a short transmembrane,
15260                                                                    and a cytoplasmic tyrosine kinase domain. D2 is one of
Polyglutamine (polyQ) diseases such as Huntington’s disease              the important subdomain in the extracellular domain. D2
are inherited neurodegenerative disorders caused by the                  domain can be produced in large quantity & in a very short


period of time by growing the Escherichia coli (E. coli) cells            of the asparagine. When the asparagine within the sequence
in the nutrient medium. But during over expression the E.                 was mutated to alanine, the time required to form fibril
coli have the tendency to accumulate the target proteins into             nuclei was extended, as determined in Thioflavin-T assay.
inactive insoluble aggregate called inclusion bodies. The                 This suggests that rearrangement reaction within an Asn-Gly
formation of inclusion bodies limits its use in understanding             sequence is involved in the early stages of GroES amyloid
the structure and function of several biologically important              fibril formation.
proteins. Therefore there is strong need for the development
of strategies to mitigate the formation of such inclusion                 104
bodies. In this study we expressed the D2 domain of FGFR
and tested the effect of gold on the formation of inclusion               Biophysical characterization of fibrillogenic fragments of
bodies using bacterial host system. We treated the E. coli                human prion protein
cells with increasing concentrations of gold (in the form of              Jason Yau1,2, Simon Sharpe1,2
potassium tetrachloroaurate (III) hydrate) and analyzed the
                                                                           Molecular Structure and Function Program, Hospital for Sick
expression of the proteins using TEM analysis, UV-Visible                 Children, 555 University Avenue, Room 3209, Black Wing,
spectrum and SDS-PAGE electrophoresis. Further we                         Toronto, M5G 1X8, Canada, 2Department of Biochemistry,
purified the soluble portion of D2 domain of FGFR using the               University of Toronto, 1 King’s College Circle, Medical
Nickel affinity column chromatography. Result of the present              Sciences Building, Room 5207, Toronto, ON, M5S 1A8,
study pave way for an in-depth investigation of the molecular             Canada
mechanism(s) underlying the activation of FGF signaling.                  The deposit of fibrillar aggregates in brain tissues is
                                                                          characteristic of many neurodegenerative disorders such
103                                                                       as Alzheimer’s and prion disease. While different proteins
                                                                          are associated with these diseases, such as Amyloid-β,
Rearrangement reaction at Asn-Gly of GroES enhances                       α-synuclein, and prion protein, they all involve misfolded
a conformation that leads to amyloid fibril formation in                  polypeptides self-assembling into β-rich fibrillar structure
early step                                                                as a pathological marker. Knowing the structural elements
Hisanori Iwasa1, Shunsuke Meshitsuka2, Kunihiro Hongo1,                   required for peptide assembly and toxicity will help us
Tomohiro Mizobata1, Yashushi Kawata1                                      understand the molecular mechanism behind the pathogenesis
  Protein Engineering, Chemistry and Biotechnology, Tottori               of amyloid diseases and other neurodegenerative disorders.
University, 4-101, Koyama-minami, Tottori, 680-8552,                      In our studies, we used fibril-forming peptides derived from
Japan, 2Integrative Bioscience, Regenerative Medicine and                 the human prion protein to study the structure of cytotoxic
Biofunction, Tottori University, 86, Nishi-machi, Yonago,                 fibrillar aggregates. Biophysical characterization by fourier-
683-8503, Japan                                                           transformed infra-red spectroscopy, electron microscopy,
Recent studies have proposed that amyloid fibrils are a                   and thioflavin T binding indicate that the peptides fibrillize
common intrinsic form of protein structure, and many                      and contain β-sheet secondary structure, but with differences
proteins that are not directly related to diseases may form               that may correlate with cytotoxicity. Fibril fragmentation
typical amyloid fibrils under specific conditions. Previously,            of two of the three peptides has been reported to increase
we found that E. coli chaperonin GroES, a cochaperonin                    toxicity in cell cultures, suggesting a role of sequence and
which works with GroEL to mediate the folding reactions                   fibril assembly on amyloid toxicity. Solid-state NMR studies
of various proteins, forms a typical amyloid fibril from a                are being carried out to determine the peptide arrangements
guanidine hydrochloride-unfolded state, and determined the                in the fibril core, and have allowed us to obtain details on
core sequence of this fibril. In this study, in order to elucidate        the modes of peptide assembly. Progress towards high-
the very early, initial conformational changes that may act as            resolution structure will be reported, and biophysical analysis
an important trigger in amyloid fibril formation of GroES,                of peptide-membrane interaction with model membrane will
we monitored GroES amyloid fibril formation using solution                be correlated with the structural properties of the amyloid
NMR spectroscopy. In real-time HSQC measurements,                         peptides. These investigations will allow us to compare
conformational changes in the contiguous amino acid                       conformational similarities between the fibrils and give us
sequence before the fibril core region were observed during               detailed insight into the role of local structure on cellular
fibril formation. 15N relaxation analysis indicated that this             toxicity.
soluble intermediate existed as a monomer. To examine the                 This project is supported by funding from PrioNet Canada
mechanism of the conformational changes, mass spectroscopy                and the National Science and Engineering Research Council
and native-PAGE experiments were performed. Interestingly,                of Canada. Dr. Simon Sharpe is a Tier II holder of Canada
it was found from the results that the conformational changes             Research Chair.
were preceded by a rearrangement of the polypeptide
backbone in a specific Asn-Gly sequence area, which yielded
beta-aspartic acids generated as a consequence of deamidation


105                                                                    The formation of amyloid, a cross-beta-sheet fibrillar
                                                                       aggregate, is associated with a variety of aging-associated
Low density lipoprotein (LDL) Inversely Modulates                      degenerative diseases. We report the existence of a mammalian
Amylin Aggregation and Toxicity in Cultured Rat Beta-                  amyloid disaggregase activity that is present in all tissues and
Cells                                                                  cell types tested. Homogenates from mammalian tissues and
Saurabh Trikha1, Aleksandar Jeremic2                                   cell lines are able to disaggregate amyloid fibrils composed
  Biological Sciences, George Washington University, 2023 G            of Abeta1-40 or the 8 kDa plasma gelsolin fragment. The
street NW, Lisner Hall Room 340, Washington, DC, 20052,                mammalian disaggregase activity is sensitive to proteinase
  Biological Sciences, George Washington University, 2023 G            K digestion and can be uncoupled from proteolysis activity
street NW, Lisner Hall, room 340, Washington, DC, 20052                using a protease inhibitor cocktail. Amyloid disaggregation
Amylin is a 37 amino acids pancreatic peptide hormone                  and proteolysis activities are remarkably resistant to
that is stored and co-secreted with insulin by islet beta-             changes in temperature and pH. Both mammalian and
cells. Under physiological conditions, amylin is found in              nematode amyloid disaggregase activities can be purified by
its soluble monomeric form. However, under pathological                fractionation, and active fractions can be identified using an
conditions associated with type 2 diabetes mellitus (TTDM),            in vitro kinetic disaggregation assay. Combining purification
amylin self-assembles into higher molecular weight species,            with Multidimensional Protein Identification Technology
oligomers and fibrils. As an increase in amylin aggregation is         allows for the identification of the molecules responsible
accompanied with high low density lipoprotein (LDL) plasma             for amyloid disaggregation, offering the possibility of
levels, a hallmark of TTDM, the objective of this study was            enhancement of this protective activity that likely guards
to determine possible modulatory effect of LDL on amylin               against aging-related, aggregation-associated diseases.
aggregation, and the extent to which amylin and LDL affect
viability of pancreatic beta-cells. To investigate amylin-LDL          107
interactions, we used rat pancreatic beta-cells (RINm5F) as a
model system. The regulatory effect of LDL on the kinetics             Probing Unique Structural Characteristics between
and extent of amylin aggregation in solution was monitored             Different Prion Disease Strains via Chemical Crosslinking
by Thioflavin-T (ThT) fluorescence assay. The formation                and Mass Spectrometry
of amylin oligomers was visualized using the dot-blot                  Michalee Moen, Michele McGuirl
(oligomer) assay and western blot. 3-(4, 5-Di methylthiaol-            Division of Biological Sciences, University of Montana, 32
2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxic                 Campus Drive, Clapp 206, Missoula, MT, 59812
assay was employed to study effects of LDL and amylin on               Prion diseases are a group of fatal neurodegenerative diseases
beta-cell viability. A significant (p<0.01, n=6; Student`s t           that include mad cow disease in cattle, chronic wasting disease
test) and dose-dependent (20-200µM) inhibition of amylin               in deer and elk, and Creutzfeldt-Jakob disease, fatal familial
aggregation by LDL was demonstrated by ThT fibrilization               insomnia, kuru and Gerstmann-Sträussler-Scheinker disease
assay. In contrast, LDL stimulated the formation of toxic              in humans. The causative agent is a misfolding event of the
amylin oligomers, as revealed using A11-oligomer specific              prion protein, which is associated with an increase of beta
immunoassay. At concentrations (20-200µM) that stimulated              sheet character within the protein, and often the formation
amylin oligomerization, LDL also potentiated amylin toxicity           of large, insoluble and fibrillar aggregates. Interestingly, the
in RINm5F beta-cells, but it had no significant effect on the          human prion diseases have discrete clinical symptoms, affect
beta-cell viability on its own (p>0.05, n=6; Student`s t test).        distinct areas within the brain, and have radically different
Our results suggest that LDL can contribute to the etiology of         ages of onset and disease durations, though they are all caused
TTDM at least in part by potentiating amylin-induced beta-             by misfolding of the same protein. Based on these unique
cell death.                                                            disease profiles, we hypothesize that there may be structural
This work was supported by the George Washington                       differences among the disease-related fibrils.
University Facilitating Fund for Research (to AJ).                     The long term goal of this study is to probe structural
                                                                       differences among the major disease strains using chemical
106                                                                    crosslinking/mass spectrometry. This technique will identify
                                                                       differences in subunit interactions that may exist in fibrils
Characterization and Identification of Amyloid                         produced from mutants associated with disease. A cysteine
Disaggregases                                                          (Cys) residue is introduced by site-directed mutagenesis,
Amber Murray1, James Solomon1, Ya-Juan Wang1, Tingwei                  the recombinant protein is purified and a benzophenone
Mu1, William Balch2, Jeffery Kelly1                                    crosslinker is coupled to the Cys while the protein is in its
  The Scripps Research Institute, 4122 Sorrento Valley Blvd            monomeric state. Fibrillization is then chemically induced
Ste 107, BCC-265, San Diego, CA, 92121, 2The Scripps                   (Apetri et al. Biochemistry (2005) 44:15880). The fibrils
Research Institute, 4122 Sorrento Valley Blvd Ste 107,                 are crosslinked with UV light and inter-subunit crosslinks
MB114, San Diego, CA, 92121                                            are identified by MS/MS analysis of the tryptic peptides.


We report here the results for mutants at positions 164, 192             109
and 219 as proof of concept, which we believe will support
the Surewicz model (Cobb et al. PNAS (2007) 104:18946)                   Mechanisms of Cu, Zn superoxide dismutase misfolding
for the fibrillar structure. Mutations associated with specific          induced by hydrogen peroxide:           Implications for
diseases will be introduced next. Funding was from the NIH               amyotrophic lateral sclerosis
via 1RO1GM081649-02 (McGuirl) and a subproject award                     Vikram Mulligan1, Priya Sharda1, Kevin Hadley2, Avijit
from P20 RR020185-01 (Harmsen, PI).                                      Chakrabartty3
                                                                          Dept. of Biochemistry, 2Dept. of Medical Biophysics,
                                                                          Depts. of Biochemistry and Medical Biophysics, University
                                                                         of Toronto, Toronto Medical Discovery Tower Rm. 4-305,
Characterization of prion protein multimers formed                       MaRS Building, 101 College Street, Toronto, ON, M5G 1L7,
under mildly acidic conditions                                           Canada
Xu Qi1, Michele McGuirl2                                                 The Cu,Zn superoxide dismutase (SOD1) is known to play
  Division of Biological Sciences, The University of Montana,            a causative role in the plurality of familial amyotrophic
CHCB 206, 32 Campus Drive, The University of Montana,                    lateral sclerosis (fALS) cases, and in a subset of apparently
Missoula, MT, 59812, 2Division of Biological Sciences, The               sporadic (sALS) cases. Misfolding and aggregation of SOD1
University of Montana, CHCB 204, 32 Campus Drive, The                    is a hallmark of SOD1-associated ALS, but the mechanisms
University of Montana, Missoula, MT, 59812                               by which such a stable protein could be induced to misfold
Conversion of prion protein from its cellular form (PrPC)                and aggregate remain unclear. There is evidence that SOD1’s
to the scrapie isoform (PrPSc) constitutes the key event                 role as a scavenger of reactive oxygen species may confer
of the etiology of a group of neurodegenerative diseases                 a heightened risk of acquiring oxidative damage that could
designated transmissible spongiform encephalopathies.                    lead to misfolding and aggregation. Here, we show that
However, the direct association between PrPSc and prion                  hydrogen peroxide, a product of the dismutase reaction
diseases pathogenesis has been increasingly challenged                   catalyzed by SOD1, induces release of bound copper and
due to the discrepancies between amyloid plaque deposits                 zinc ligands by a four-state branched mechanism involving
and clinical symptoms. Accumulating evidence suggests                    sequential oxidation of histidine residues that bind the metal
that amyloid-associated pathologies are caused by transient              ions. Although peroxide treatment does not cause global
toxic intermediates that eventually convert to relatively inert          unfolding, it does trigger partial dissociation of the SOD1
amyloid deposits.                                                        dimer. The copper-deficient, zinc-deficient, and monomeric
For other types of amyloid deposits, it has been shown that              species produced by oxidative damage are key candidates
the fibrils are in a slow dynamic equilibrium with the soluble           for early species in the SOD1 misfolding and aggregation
peptides. Inspired by this and the fact that PcPSc enters                cascade, and could be useful targets for drugs intended to
lysosomes once it is recycled from the cell surface, we have             block progression of SOD1-associated ALS.
investigated the effect of mildly acidic pH on the stability of          This research was funded by the Canadian Institutes of Health
recombinant PrP fibrils as models of PrPSc. We found that                Research, and by the ALS Society of Canada.
the fibrils dissociate to a mixture of soluble monomeric,
oligomeric and multimeric species under mildly acidic                    110
conditions. Asymmetric flow-field flow fractionation analysis
suggests the major species has an apparent molecular weight              In vitro and In vivo Aggregation of Mutants of a β-Clam
about 4 MDa, which corresponds to 200 monomers. We                       Protein
propose that this species might be the toxic multimers formed            Mylene Ferrolino1, Anastasia Zhuravleva2, Zoya Ignatova3,
transiently from the monomeric species during fibrillation,              Amanda Clouser2, Lila Gierasch2
which then convert to fibrils. Circular dichroism and fourier
                                                                          Molecular and Cell Biology, 2Biochemistry and Molecular
transform infrared (FT-IR) spectra show that the multimers               Biology, University of Massachusetts Amherst, 710 North
adopt a β-sheet structure. FT-IR spectra also suggest a more             Pleasant St., Amherst, MA, 01002, 3Institute of Biology and
flexible intermolecular β-sheet structure. Electron microscope           Biochemistry, University of Potsdam, Karl-Liebknecht-Str.
reveals that in contrast to the mature fibrils, the multimers are        24-25, Haus 25, Potsdam-Golm, Germany
much shorter. Moreover, the multimers are more efficient at              Protein aggregation has been implicated in a wide variety of
propagating amyloid formation than the fibrils. We propose that          neurodegenerative diseases and its complexity has become
the multimers have more active ends as compared with fibrils,            a major challenge in the development of therapeutics. The
which may accelerate the fibrillation process. This research             tendency of proteins to form aggregates has been quite well
is supported by NIH 1RO1GM081649-02 (McGuirl) and a                      characterized in defined media but is poorly understood in
subproject award from P20 RR020185-01 (Harmsen, PI).                     the complex intracellular environment. We are interested
                                                                         in developing approaches to tackle these challenges using
                                                                         a predominantly β-sheet protein whose in vitro folding has


been explored in detail: cellular retinoic acid binding protein            surfaces tend to be aggregation-prone. It may be that
I (CRABP 1) and E. coli as a cellular model. Interestingly,                evolution avoids aggregation-prone sequences to prevent not
despite their β-barrel structure, there are no amyloid-based               only toxic gain of function, but also the loss of function of
diseases associated with proteins from the intracellular                   highly-interactive, functionally important proteins. At nearly
lipid binding protein family to which CRABP 1 belongs.                     identical average expression levels, essential proteins have
Nonetheless, we have identified many point mutations                       a lower average aggregation propensity than non-essential
that increase the aggregation propensity of CRABP 1. We                    proteins. In addition, expression and aggregation propensity
are interested in determining the mechanism that drives                    of E. coli transcription factors decrease as the number of gene
aggregation in these CRABP 1 variants both in vitro and in                 targets increases. Having identified loss-of-function pressure
vivo. We hypothesize that aggregation propensity may be                    on solubility, we investigated whether cellular parameters
increased by destabilization of the protein, by stabilization              could shift the solubility edge. We observed separate edges
of an aggregation-prone intermediate, or by the creation of                for essential proteins and for non-essential proteins with
a new aggregation-prone region. Our goal is to compare the                 ontologies associated with high aggregation propensities.
nature of the cores of in vitro- and in vivo-derived aggregates            This indicates that the solubility requirements are not static
from different CRABP 1 variants. We are using DMSO                         and universal, but that they shift based on proteins’ functional
quenched hydrogen-deuterium exchange NMR (HDX NMR)                         importance. These requirements may also shift under the
as a method to identify the aggregation cores at a residue                 distinct conditions of stress and disease.
specific level. We have found that inclusion bodies from                   PC is supported by a Fulbright Scholarship.
an aggregation-prone CRABP I mutant, F71A contain short
aggregation cores, which correlate with predicted aggregation
hotspots. Future identification of core sequences of other                 Applications of Mass Spectrometry
mutants will provide us insights on what drives proteins to
aggregate outside and inside the cell.                                     to Biological Problems (112 – 115)
111                                                                        112
Shifting the edge: Protein function determines protein                     Possibilities and pitfalls in quantifying the extent of
solubility requirements                                                    cysteine sulfenic acid modification of specific proteins
Prajwal Ciryam1,2, Gian Gaetano Tartaglia1, Michele                        within complex biofluids
Vendruscolo1, Christopher M. Dobson1                                       Douglas Rehder, Chad Borges
  Department of Chemistry, University of Cambridge,                        The Biodesign Institute / MB, Arizona State University, P.O.
Lensfield Rd, Cambridge, CB21EW, United Kingdom,                           Box 876601, Tempe, AZ, 85287
  Department of Biochemistry, Molecular Biology, and                       Cysteine sulfenic acid (Cys-SOH) plays important roles in
Cell Biology, Northwestern University, 2205 Tech Drive,                    the redox regulation of numerous proteins. As a relatively
Evanston, IL, 60208                                                        unstable posttranslational protein modification it is difficult
A fundamental requirement for life is that proteins remain                 to quantify the degree to which any particular protein
soluble in the cell. When protein homeostasis fails to maintain            is modified by Cys-SOH within a complex biological
solubility, toxic protein aggregates can form, causing                     environment. The goal of these studies was to move a step
Alzheimer’s, Parkinson’s, and other diseases. It has been                  beyond detection and into the relative quantification of Cys-
observed that the solubility requirement of proteins forms an              SOH within specific proteins found in a complex biological
“edge” in which maximum expression level anti-correlates                   setting—namely, human plasma. This presentation will
with aggregation propensity. Proteins occupy a space on the                describe the possibilities and limitations of performing
edge between solubility and insolubility. In human cells, this             such analyses based on the use of thionitrobenzoic acid and
process is dependent on subcellular localization. This suggests            dimedone-based probes which are commonly employed to
that 1) there is evolutionary pressure to maintain proteins                trap Cys-SOH. Results obtained by electrospray ionization-
solubility at a critical level and 2) this pressure is modulated by        based mass spectrometric immunoassay reveal the optimal
a protein’s environment. We investigated cellular parameters               type of probe for such analyses as well as the reproducible
that may affect proteins’ solubility requirements. For 1889                relative quantification of Cys-SOH within albumin and
E. coli proteins and 2681 human proteins, we predicted                     transthyretin extracted from human plasma—the latter as a
aggregation propensities using the Zyggregator method;                     protein previously unknown to be modified by Cys-SOH. The
obtained mRNA expression levels for corresponding genes                    relative quantification of Cys-SOH within specific proteins
from the literature; and obtained binary protein interaction data          in a complex biological setting can be accomplished, but
from the Human Protein Reference Database. As interaction                  several analytical precautions related to trapping, detecting,
number increases, aggregation propensity decreases and                     and quantifying Cys-SOH must be taken into account prior to
expression increases. This is surprising, because interaction              pursuing its study in such matrices.


113                                                                     114
Proteomic analysis of 3t3-LI adipocyte mitochondria                     Mass Spectrometry Reveals a Subunit Interaction Map of
during differentiation, maturation, and hypertrophic                    the Phosphorylase Kinase Complex
enlargement.                                                            Owen Nadeau1, Laura Lane2,3, Carol Robinson4, Gerald
William Russell1, Stephanie Cologna1, Billy Newton2, Arul               Carlson1
Jayaraman2, David Russell1                                              1
                                                                         Biochemistry, Univ. of Kansas Medical Center, 3901
  Chemistry, Texas A&M University, Department of Chemistry,             Rainbow Blvd., Kansas City, KS, 66103, 2Chemistry, Univ.
Texas A&M University, MS 3255, College Station, TX, 77843,              of Cambridge, Lensfield Road, Cambridge, United Kingdom,
  Chemical Engineery, Texas A&M University, Department of               3
                                                                         Chemistry, Univ. of Oxford, Mansfield Road, Oxford, United
Chemical Engineering, Texas A&M University, Mail Stop                   Kingdom, 4Chemistry, University of Oxford, South Parks
3122, College Station, TX, 77843                                        Road, Oxford, United Kingdom
Several harmful metabolic and inflammatory markers are                  Phosphorylase kinase (PhK) is a 1.3 MDa (αβγδ)4 complex
known to be associated with obesity. This disease is not simply         that regulates glycogenolysis in skeletal muscle. The
a problem of excess energy storage, but is characterized as             activity of the catalytic γ subunit is regulated by allosteric
metabolic dysregulation of adipose tissue. Studies of adipose           activators that target its regulatory α, β and δ subunits. The
tissue have shown that the largest and most lipid-laden,                approximate locations of the subunits have been mapped by
(i.e. hypertrophic adipocytes) produce the highest levels of            immunoelectron microscopy of the complex, showing it to
inflammatory chemokines. Due to the roles of mitochondria               comprise two large lobes that associate with D2 symmetry
in energy production and metabolism we sought to conduct                through interconnecting bridges. Within the lobes, the α, β,
a proteomic investigation to identify protein expression                γ and δ subunits pack head to head as protomeric tetramers
changes in adipocyte mitochondria as adipocytes age and                 to form (αβγδ)2 dimers; however, little else is known about
accumulate lipids. We have studied changes to the adipocyte             the interactions of these subunits within the PhK complex.
mitochondria during differentiation through the hypertrophic            Structural information regarding subunit interactions in the
state.                                                                  complex has mostly come from bottom-up analyses by MS
Quantitative proteomics were conducted using an iTRAQ                   of crosslinked inter-subunit digests. To complement our
6-plex kit, where mitochondrial proteins from undifferentiated          crosslinking approach, we have recently employed top-down
3T3-l1 preadipocytes and adipocytes that upon 0, 4, 7, 10, 14,          MS methods designed to study the organization of large
and 18 days post-differentiation were each labeled, digested,           protein complexes. We show for the first time mass spectra of
and subjected to two dimensions of separation followed by               intact, nonactivated PhK, as well as its counterpart activated
LC-MALDI-TOF MS/MS analysis.                                            by phosphorylation. Native and phosphorylated complexes
                                                                        exhibit different dissociation patterns, forming αγδ and γδ
Preliminary results from this study indicate that we are able to        subcomplexes with the former and a β4 subcomplex with
identify 3972 unique peptides corresponding to 387 proteins             the latter. The first two subcomplexes have been previously
(99% confidence level) from adipocyte mitochrondia.                     observed by crosslinking and other techniques, thus validating
From this set of proteins, we found that the majority of                this approach. The presence of the previously unobserved
enzymes and transporters involved in the TCA cycle, fatty               β4 subcomplex supports our previous hypothesis that the
acid oxidation, and ATP synthesis were up-regulated upon                PhK complex packs as a bridged core of four β subunits
differentiation, and maintain or increase these levels through          upon which four αγδ trimers are arrayed. Our initial results
a state of adipocyte hypertrophy. These results suggest that            demonstrate that this approach should prove invaluable in
cultured adipocytes enter a state of metabolic overdrive                further comparing subunit interactions in nonactivated and
where increased flux through the TCA cycle and increased                activated forms of PhK (Supported by NIH grant DK32953,
fatty acid oxidation occur simultaneously. We also were able            the Royal Society and the Engineering and Physical Science
to observe an increase in the concentration of reactive oxygen          Research Council).
species (ROS) from separate studies which suggests that
this increase likely resulted in a concurrent increase in anti-
oxidant and detoxification enzymes such as peroxiredoxins 3
and 5, carbonyl reductase and theoredoxin sulfurtransferase.
Detailed analysis of this data will be presented.


115                                                                  Computational Biology (116 – 120)
Chemical Cross-linking and Mass Spectrometry for
Structural Analysis of the Metal-Resistance Protein CusB             116
and Determination of the CusB/CusF Interaction Site                  Rigorous Approximation of Continuum Electrostatics:
Tiffany Mealman, Vicki Wysocki, Megan McEvoy                         The Impact of Fast Algorithms and GPU Computing
Chemistry & Biochemistry, University of Arizona, 1041 E.             Jaydeep P. Bardhan1, Rio Yokota2, Matthew G. Knepley1,3,
Lowell St., Tucson, AZ, 85721                                        Lorena A. Barba4
The E. coli Cu(I)/Ag(I) efflux system, CusCFBA, confers              1
                                                                      Department of Molecular Biophysics and Physiology, Rush
metal resistance by exporting toxic metal ions out of the            University Medical Center, 1750 W. Harrison St, 12th floor,
periplasm. CusF, a novel periplasmic metallochaparone,               Jelke Southcenter Building, Chicago, IL, 60612, 2Department
is involved in direct metal transfer with the periplasmic            of Mathematics, University of Bristol, University Walk, BS8
membrane fusion protein (MFP), CusB. Metal binding at                1TW, Bristol, United Kingdom, 3Computation Institute,
the N-terminus of CusB causes a conformational change in             University of Chicago, Searle Chemistry Laboratory, 5735
the protein that has yet to be determined. Chemical cross-           South Ellis Avenue, Chicago, IL, 60637, 4Department of
linking coupled with mass spectrometry is an advantageous            Mechanical Engineering, Boston University, 110 Cummington
technique because it allows protein structure interactions to        St, Boston, MA, 02215
be resolved at the amino acid level. In this work, chemical          The computational challenges associated with evaluating
cross-linking and mass spectrometry were used to: 1) identify        electrostatic interactions within and between biomolecules
the interaction site of CusF/CusB, 2) map the conformational         and surrounding solvent are well known. In this poster we
change of CusB upon metal binding, 3) gain structural                describe an open-source and parallel implementation of the
information about the N-terminus of CusB.                            BIBEE (boundary-integral-based electrostatics estimation)
Proteins were cross-linked in vitro using 7.7 Å and 11.4 Å           model for molecular electrostatics, which uses the PetFMM
homobifunctional isotope-labeled amine reactive cross-               library for a scalable and GPU-accelerated fast multipole
linkers (BS2G-d0/d4 and BS3-d0/d4). Tryptic digests were             method. This solver will enable the detailed, rigorous
performed and the reaction mixtures analyzed with nano-              analysis of large macromolecular systems that previously
HPLC coupled to an LTQ-Orbitrap (Thermo) or a DecaXP                 could only be studied using coarse-grained representations
Plus Ion Trap (Thermo). The most abundant peaks in an                or heuristic electrostatic models. BIBEE methods are
MS survey scan were selected for tandem MS using the data            mathematical approximations to the mixed-dielectric linear
dependent scanning mode. Data analysis was performed                 continuum model popularized in solvers like APBS and
using Phenyx, Popitam, and manual analysis with Protein              DelPhi. The rigorous link between the approximate and
XXX for MS/MS fragment ion confirmation.                             actual electrostatic model gives BIBEE numerous advantages
Cross-linking results confirm that the interaction between           over heuristics like the generalized-Born (GB) models. For
CusF and CusB is metal-dependent, with two cross-linked              example, we have been able to prove analytically that variants
peptides identified between CusF and CusB. The same                  of BIBEE generate electrostatic free energies that are upper
sequence of CusB was identified in both cross-links and is           and lower bounds to the actual free energy from solving the
located in the N-terminal metal binding region. The CusF             mixed-dielectric Poisson problem. Computational results
peptides participating in the cross-links are adjacent to one        show speedups on two levels: first, BIBEE can estimate
another and compose the metal binding face of CusF. Our              electrostatic free energies of protein--protein complexes an
results show that CusF and CusB interact at the metal-               order of magnitude faster than full simulation. Second,
bound regions. Intramolecular CusB cross-linking results             general-purpose graphics processing units (GPU) exhibit
are improving structural information, particularly in the            orders of magnitude speedup over traditional CPU clusters.
N-terminal region.
NIH GM079192


117                                                                    and coarser libraries depending on sampling needs. Here we
                                                                       present an enhancement in the design of a conformer library,
Mechanism of sliding clamp opening by the clamp loader                 by introducing the sorting of its conformers based on their
RFC: Insight from atomistic simulations                                efficiency in fitting natural protein environments that contain
Ivaylo Ivanov1, J. Andrew McCammon2, John Tainer3                      the same amino acid type. We have produced a very extended
  Chemistry, Georgia State University, P.O. Box 4098,                  conformer library and measured the energies of each member
Atlanta, GA, 30302, 2University of California – San Diego,             in a series of positions in protein crystal structures. We have
9500 Gilman Drive, La Jolla, CA, 92093-0365, 3The Scripps              then sorted the conformers in order of their propensity to fit
Research Institute, 10550 North Torrey Pines Road, La Jolla,           the most environments with the lowest energy. The result
CA, 92037                                                              is a library that is very efficient in repacking side chains
PCNA is a ring-shaped protein (sliding clamp) that can                 even at small sampling. The library also provides maximum
encircle and slide along DNA. Recently, groundbreaking                 flexibility, allowing a user to tailor the library size to the
structural biology work has provided a glimpse into the                specific needs of the problem, and to distribute sampling
workings of the cellular machinery responsible for opening             differentially according to the nature of the environment
and loading sliding clamps onto DNA and opened up the                  of each position (for example, solvent exposed, buried, or
possibility of investigating clamp-loader assemblies in atomic         intermediate). Together the enhanced performance and the
detail. Herein we present results from atomistic simulations           added flexibility can improve significantly the accuracy of
of the initial steps in the clamp-loading cycle. We have used          side chain prediction in docking, modeling and design.
molecular dynamics to obtain an atomistic model of the
complex of the clamp loader RFC with an open clamp. The                119
out-of-plane twisting of the open PCNA led to a right-handed
helical conformation and the formation of an extended                  Binding Free Energy Analysis based on GPGPU cluster
interface with RFC. We characterized this interface in terms           Masakazu Sekijima1, Kiyotaka Misoo2, Kazuki Ohno3,
of residue contacts, electrostatic and shape complementarity.          Masaya Orita3
Comparison of free energy profiles for clamp opening in the
                                                                        Global Scientific Information and Computing Center, Tokyo
presence and absence of RFC allowed us to substantiate the             Institute of Technology, 2-12-1-i7-5, Ookayama, Meguro-ku,
role of RFC in the initial stage of the clamp-loading cycle.           TOKYO, 1528550, Jordan, 2Information and Mathematical
Additionally, low-resolution elastic network modeling                  Science Laboratory Inc., Meikei Bldg. 1-5-21, Ohtsuka,
revealed the global motions of the PCNA/RFC assembly and               Bunkyo-ku, TOKYO, 1120012, Japan, 3Astellas Pharma Inc.,
provided insight into the camp-opening process.                        21, Miyukigaoka, Tsukuba-Shi, TOKYO, 3058585, Japan
                                                                       Molecular dynamics (MD) simulations are widely used
118                                                                    for simulating molecular motion for gaining a deeper
                                                                       understanding of chemical reactions, fluid flow, phase
An energy-sorted conformer library improves side chain                 transitions, and other physical phenomena due to molecular
sampling in protein modeling and design                                interactions.
Alessandro Senes1, Sabareesh Subramaniam2
  Dept of Biochemistry, University of Wisconsin-Madison,               General Purpose Graphics Processing Unit (GPGPU) is
433 Babcock Dr, Madison, WI, 53706, 2Dept of Computer                  recently applied for High Performance Computing (HPC).
Sciences, University of Wisconsin-Madison, 433 Babcock                 We can obtain rapidly increasing computational power from
Dr, room 419, Madison, WI, 53706                                       GPGPU cluster.

The prediction of side chain conformation is an important              We optimized MD simulation program on GPGPU cluster.
element in protein design, docking and both de novo and                In our presentation, we will show performances of some
homology-based structural modeling. In most cases these                proteins. On the other hand, we developed the binding free
applications sample side chain flexibility according to rotamer        energy calculation system based on molecular dynamics
libraries, which are representative orientations selected to           simulation. The binding free energy is important information
approximate the conformational space that each amino acid can          for understanding interactions between protein and protein or
adopt in natural proteins. The most commonly used rotamer              protein and chemical compounds. We also show calculation
libraries derive from the statistical distribution of the side         results of the binding free energy of some targets.
chain’s dihedral angles as observed in the structural database.
An alternative is offered by “conformer” libraries, which are
collections of actual side chain conformations extracted from
protein crystal structures. Conformer libraries introduce
natural variation of all degrees of freedom (including angles
and bond distances and not limited to dihedrals), which can be
beneficial. They can also be easily adapted to produce finer


120                                                                     appears as dimers. The effect of dimerization in the cellular role
                                                                        of talin is the subject of this work. We show that monomeric
Inter-chain Disulfide Scrambling in Human IgG2                          talin fails to localize at the focal adhesion sites. It impacts
Antibodies: An Atomistic Insights from Molecular                        the spreading and adhesion of cells, which initially spread
Dynamics Simulations                                                    but later on retract and can not form proper focal adhesion
Xiaoling Wang, Satish Singh, Sandeep Kumar                              complexes. Cells containing only monomeric talin fail to
Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield,               apply forces on the extracellular matrix (ECM) and have
MO, 63017                                                               decreased their ability to show reinforcement as a response to
Abstract                                                                the application of external forces. The oligomerisation state of
Human IgG2 mAbs have been reported to undergo disulfide                 talin also affects the level of phosphorylation of phosphatases
scrambling in vivo. Several distinct but inter-convertible              involved in the focal adhesion complexes. If cells do not
disulfide isoforms have been characterized experimentally. We           spread properly, can not apply forces on their substrates and
have performed all-atom explicit water molecular dynamics               do not communicate the ECM with the cytoskeleton, their
simulations to study hinge-region dynamics. Our model                   proliferation, adhesion, and migration patterns will be severely
system (Hinge++) is a mouse IgG2a antibody crystal structure            impaired. These results have outstanding implications for
with humanized hinge region. Only the hinge region and the              biological and medical research.
adjoining domains, namely, CH1, CL and CH2 were simulated.
Five independent simulations have been performed. These
include one control simulation where all cysteine residues were
                                                                        Enzyme Kinetics and Mechanism
disulfide bonded as in the canonical IgG2-A form. Of the other          (122 – 128)
four simulations, two had all Cys residues reduced, one had
the twelve Cys residues forming all six inter-chain disulfide           122
bonds reduced, and one had eight Cys residues forming four
inter-chain disulfide bonds reduced. All simulations show               Allosteric communication between subunits of the dimeric
that radius of gyration of Hinge++ decreases over simulation            phosphofructokinase-2 of E. coli analyzed by hybrid
time and closer inter-domain contacts are formed across the             enzymes
hinge-region. The Sulphur – Sulphur atom distances show                 Jorge Babul, Mauricio Baez
large fluctuations in case of the Cys residue pairs that were           Facultad de Ciencias, Universidad de Chile, Las Palmeras
involved in the canonical inter-chain disulfide bonds. In               3425, Santiago, Chile
contrast, these distances are relatively stable for Cys residue         Phosphofructokinase-2 (Pfk-2) shows allosteric inhibition by
pairs that were involved in the intra-chain disulfide bonds.            its substrate MgATP, a regulation important for avoiding a
Closer inter-domain contacts, looser inter-chain packing in             futile cycle of ATP hydrolysis under gluconeogenic growth
the hinge region and high mobility of inter-chain disulfide             conditions. In this work, subunit-hybrid enzymes of mutants
forming Cys residues could be the early events in human IgG2            of homodimeric Pfk-2 were created and studied in an
disulfide scrambling. In particular, our simulations indicate           examination of the mechanism of allosteric inhibition by ATP.
that C-terminal Cys residue in the light chain (C214) could             Kinetics studies of ATP inhibition indicate that the nucleotide
approach heavy chain Cys residues in the upper hinge region.            shifts the saturation for fructose-6-P from hyperbolic to
This could initiate disulfide scrambling. Interestingly, contact        sigmoid curves, together with a decrease in the apparent
map analyses indicate that hinge region Cys residues could              affinity for fructose-6-P. Sigmoidal substrate binding induced
come in frequent contact with the Cys residues other than their         by an allosteric inhibitor is a feature predicted by specific
canonical disulfide partners. Overall, these simulations have           models of mechanisms that involve allosteric communication
led to better theoretical understanding of the hinge region and         among binding sites in oligomeric proteins. Since the dimeric
inter-chain disulfide pairing dynamics in human IgG2 mAbs.              Pfk-2 structure shows an ATP allosteric site in each subunit,
                                                                        the negative interplay between fructose-6-P and ATP could
                                                                        be originated in each subunit. However, the allosteric ATP
Cytoskeleton (121)                                                      and fructose-6-P make several contacts with side chains
                                                                        provided by the adjacent subunit, providing structural
121                                                                     support for the apparent cooperativity of fructose-6-P. Hybrid
Role of dimerization in talin function                                  dimers containing an intact allosteric and catalytic site in
Armando del Rio, Pere Roca-Cusachs, Michael Sheetz                      one subunit only still show substrate inhibition and apparent
Biological Sciences, Columbia University, 1212 Amsterdam                sigmoidal responses for fructose-6-P. Conversely, hybrids
Avenue, new york, NY, 10027                                             dimers containing a single intact allosteric and catalytic
                                                                        site but in different subunits show attenuated inhibition and
The cytoskeletal protein talin is a key player in the process           cooperativity for fructose-6-P. These results indicate that
of cell proliferation, adhesion and migration. In cells, talin


allosteric communication between subunits would not play a               124
role in the ATP inhibition of Pfk-2. (Fondecyt 1090336).
                                                                         Probing the Active Site of Cystathionine beta-Lyase.
123                                                                      Pratik Lodha1, Susan Aitken2
                                                                          Biology, Carleton University, 209 Nesbitt Building, Carleton
Revisiting the Catalytic Mechanism of Triosephosphate                    University, 1125 Colonel By drive, ottawa, ON, K1S 5B6,
Isomerase Using Site Specific Mutants                                    Canada, 2Biology, Carleton University, 209 Nesbitt Building,
Moumita Samanta, Padmanabhan Balaram                                     Carleton University, 1125 Colonel By Drive, ottawa, ON,
Molecular Biophysics Unit, Indian Institute of Science,                  K1S 5B6, Canada
Indian Institute of Science, Bangalore, 560012, India                    Cystathionine β-Lyase (CBL) is a member of the γ-subfamily
Studies on triosephosphate isomerase have been central to                of Fold type I of pyridoxal 5’-phosphate (PLP) enzymes
the development of mechanistic enzymology. In Knowles’                   and catalyzes the hydrolysis of L-cystathionine (L-Cth)
mechanism, the interconversion of dihydroxyacetone                       via a β-elimination reaction. A series of 15 site-directed
phosphate (DHAP) and glyceraldehydes-3-phosphate, involves               mutants of 10 active-site residues of E. coli CBL (Y56,
the participation of the neutral imidazole ring of His95, as an          R58, R59, Y111, D116, Y238, Y338, S339, W340 and
acid (electrophile), despite the high pKa anticipated for the            R372) was investigated. Their effect on the kcat/Km L-Cth of the
formation of an imidazolate anion. The availability of over              β-elimination reaction varies from a reduction of only 3-fold
500 sequences from diverse organisms permits definition of               (D116A and D116N) to as much as 6 orders of magnitude
completely conserved residues, K12, T74, H95, E97, C126,                 (R372A and R372L). Comparison of the kinetic parameters
E165, P166, G208 and G228 lying proximal to the active                   for L-Cth hydrolysis with those for inhibition of E. coli
site, suggesting that evolutionary selection is constrained by           CBL by aminoethoxyvinylglycine demonstrates that residue
the need to preserve optimal catalytic function. The crystal             R58 tethers the distal carboxylate group of the substrate
structure of the yeast enzyme complexed to DHAP (1NEY)                   and confirms that residues W340 and R372 interact with
reveals that four fully conserved residues, E165, H95, E97               the a-carboxylate moiety. The order of importance of these
and K12 are oriented towards the C1 and C2 atoms of the                  residues for the activity of E. coli CBL is: R372 > S339 ≈
substrate, suggesting that E97 may be critical for catalysis. The        Y111 > Y56 ≈ R58 > Y238 > W340 ≈ R59 > Y338 ≈ D116.
site-directed mutants E97Q and E97D have been constructed                The effect of pH was also investigated and the increase in the
using Plasmodium falciparum triosephosphate isomerase                    pKa of the acidic limb and decrease in the pKa of the basic
as the template. Kinetic analysis reveals a kcat reduction by            limb of the kcat/Km L-Cth versus pH profiles of the R58K and
1000fold for the E97Q, while 100fold for E97D. The E97                   R58A mutants, respectively, support a role for this residue in
carboxylic acid side chain bridges the ε ammonium group                  modulating the pKa of an active-site residue.
of K12 and the imidazole group of H95. A mechanism of
proton transfer is presented involving the ionizable E97 side            125
chain, which permits H95 to convert between the neutral and
positively charged forms of imidazole, thus eliminating the              Mutation analysis of the violaxanthin de-epoxidase
need to involve ionization to an imidazolate. This proposal              identifies substrate binding site and residues involved in
circumvents the need to consider a mechanism which “runs                 catalysis
counter to the prejudices of mechanistic chemistry” (Lodi                Giorgia Saga1, Alejandro Giorgetti2, Christian Fufezan3,
et al,1991). C126 is proximal to the active site but does                Giorgio M. Giacometti4, Roberto Bassi2, Tomas
not directly interact with the substrate. Kinetic analysis of            Morosinotto5
the mutants (C126S, C126A, C126V, C126M and C126T)
                                                                          Biology, University of Padova, via Ugo Bassi58/B, Padova,
reveals about 10 fold drop of kcat suggesting that this residue          30170, Italy, 2Dipartimento di Biotecnologie, Università di
may be involved in maintaining optimal dynamic flexibility               Verona, Strada le Grazie 15, 37134 Verona, Italy, Verona,
of the active site residue E165.                                         Italy, 3Institut für Biochemie und Biotechnologie der
                                                                         Pflanzen3, Westfälische Wilhelms-Universität Münster,
Research support: Council of Scientific and Industrial                   Hindenburgplatz 55, Münster, Germany, 4Dipartimento di
Research (CSIR), Govt. of India.                                         Biologia, Università di Padova, Via Ugo Bassi 58 B, Padova,
                                                                         Italy, 5Dipartimento di Biologia, Università di Padova, Via
                                                                         Ugo Bassi 58 B, 35121 Padova, Italy, Padova, Italy
                                                                         Plants are exposed to variable environmental conditions
                                                                         and evolved the capability of modulating light harvesting
                                                                         efficiency in response to different light intensities. One of the
                                                                         main mechanisms of response to high light is the activation
                                                                         of xanthophyll cycle by which the carotenoid violaxanthin
                                                                         is converted to zeaxanthin by Violaxanthin De-Epoxidase


(VDE). Activation of this enzyme is triggered by acidification         (100SGDQRQVDLIP110) reveal that this shorter sequence with
of the lumen occurring in excess illumination. The ligands,            a basic residue at the P-2 position exhibits weaker binding.
violaxanthin and ascorbic acid, were docked onto the crystal           Further improvements in kinetics can occur by extending the
structure of the VDE lipocalin domain (VDEcd) obtained at pH           FbnA peptide to (100SGDQRQVDLIPKKAT114). The 11KK12
5. And in order to assess experimentally the validity of the in        segment at the P-9, P-10 positions may be acting as an extra
silico model, we identified all residues potentially involved          anchor point similar to what has been observed with the K12
in the enzymatic activity by selecting those located within            of α2AP. As with the other peptides, thrombin activatable
6 Å distance from the violaxanthin molecule. Residues so               fibrinolysis inhibitor (1FQSGQVLAALPRTSR15) has only
identified were tested for their involvement in the catalytic          one reactive glutamine. The weaker kinetic properties of
mechanism by site directed mutagenesis. Experimental                   this peptide may be due to the second Q being shifted to the
results allowed us to confirm the proposed substrate binding           P-3 position and/or due to the lack of a K-containing anchor
sites and identify two residues, D177 and Y198, which are              segment. Further knowledge on amino acid character and
fundamental for enzyme activity and suggested to participate           substrate position will be important for elucidating the
in the catalytic mechanism. All changes in charge/polarity             sources of FXIII specificity. (NIH HL068440)
of these residues caused a complete loss of activity. For
other two residues W179 and Y214 the activity was highly               127
reduced but small amounts of zeaxanthin were still produced.
An additional group of mutants showed strong effects on                Interaction of the Prolyl Isomerase Pin1 with a Multi-
activity, with residual activity around 35% of the WT or less,         Phosphorylated Substrate
all remaining mutants had activity in the range of 50-75%              Alexander Greenwood, Soumya De, Linda Nicholson
of WT.                                                                 Molecular Biology and Genetics, Cornell University,
                                                                       Biotechnology Building, Tower Road, Ithaca, NY, 14850
From these experimental data and structural information we
were able to propose a catalytic mechanism for the enzyme              The peptidyl-prolyl isomerase Pin1 plays important roles in
activity.                                                              diverse cellular processes, including the immune response.
                                                                       Pin1 is a modular enzyme, comprised of two domains: a
126                                                                    WW domain and a PPIase domain, separated by a flexible
                                                                       linker. The catalytic PPIase domain is responsible for the
Examining FXIII Specificity by Assessing the Kinetic                   isomerization of phosphorylated Ser/Thr-Pro peptide bonds.
Contributions of Individual Residues in Glutamine-                     The WW domain, a small proline recognition module, binds
Containing Substrates                                                  this same phosphorylated Ser/Thr-Pro motif but does not
Prakash Doiphode, Muriel Maurer                                        isomerize it. The Pin1 WW domain is thought to be primarily
Chemistry, University Of Louisville, 2320 South Brook                  involved in substrate recognition and cellular localization.
Street, Louisville, KY, 40292                                          In Pin1’s intracellular roles, both domains are usually both
Factor XIII (FXIII) is a transglutaminase that catalyzes               necessary. The binding mode of a dual-phosphorylated ligand
the formation of gamma-glutamyl-epsilon-lysyl crosslinks               to Pin1 was investigated using NMR spectroscopy. Peptides
in the fibrin network. Although FXIIIa targets numerous                derived from a known Pin1 substrate with two recognition
physiological substrates, there is no clear consensus                  motifs ten residues apart were synthesized. The binding of the
sequence. Glutamine-containing peptides based on α2-                   mono- and bi-phosphorylated forms of these peptides to Pin1
antiplasmin (α2AP) and S. Aureus fibronectin binding A                 or its individual domains was investigated by NMR titration
(FbnA) serve as effective models for probing the roles of              experiments. Nitrogen ZZ (NZZ) and ROESY exchange
individual amino acids in FXIII substrates. A MALDI-TOF                spectroscopy were performed to measure the Pin1-catalyzed
based kinetic assay is being employed that directly follows            rates of cis-trans isomerization at each of the two sites, with
loss of reactive substrate and formation of a cross-linked             or without a phosphate at the second site. Preliminary results
product. A Q-containing peptide interacts with FXIIIa and              are suggestive of a bivalent binding mode, which has not
ammonia becomes released. Glycine ethylester then serves               previously been demonstrated for Pin1 with a biologically
as the lysine-like substrate to complete the reaction. Loss            relevant ligand. A mutagenic approach is in progress to clarify
of substrate is followed as a function of quenched time                the impact of inter-domain association in Pin1 in the process
points. Studies with the α2-antiplasmin sequence α2AP                  of substrate recognition.
(1NQEQVSPLTLLKLGN15) indicate that Q2 at the P1 position
is reactive and the Q4 at the P-1 position plays a role in
promoting substrate binding. A K12R substitution at the P-10
position leads to improvements in binding within the active
site region but the substrate is not as well oriented for the
transglutaminase reaction. Kinetic assays with S. Aur (FbnA)


128                                                                    folding, localization, and thus function of target protein in
                                                                       vivo because of their small sizes. Moreover, these chemical
Elucidation of the Pin1-APP interaction using NMR                      reporters could serve as reaction portals where a multitude of
lineshape analysis                                                     imaging modality can be attached via a growing repertoire
Soumya De1, Evgenii Kovrigin2, Linda Nicholson1                        of bioorthogonal reactions. We have recently reported a
  Molecular Biology and Genetics, Cornell University,                  photoinduced tetrazole-alkene 1,3-dipolar cycloaddition
241 Biotech Building, Cornell University, Ithaca, NY,                  reaction (“photoclick chemistry”) that can be employed to
14853, 2Biochemistry, Medical College of Wisconsin, 8701               functionalize proteins selectively both in biological medium
Watertown Plank Road, Milwaukee, WI, 53226                             and in living cells. Detailed studies of this reaction revealed a
The peptidyl-prolyl isomerase Pin1 catalyzes the cis-trans             broad reaction scope, an excellent solvent compatibility, and
isomerization of the phosphorylated Thr668-Pro669 peptide              a tunable tetrazole photoactivation wavelength. Furthermore,
bond in the amyloid precursor protein (APP). This activity             fast reaction rates were obtained when the HOMO
regulates the proteolytic processing fate of APP and thus, is          energies of the nitrile imine dipoles are lifted through the
implicated in playing a crucial role in Alzheimer’s disease.           substituent effect. In this presentation, we wish to report the
We are employing NMR spectroscopy and isothermal titration             development of alkene amino acids such as homoallylglycine
calorimetry (ITC) to elucidate the Pin1-APP interaction                as bioorthogonal chemical reporters, and in conjunction of
and its impact on Alzheimer’s disease. Interaction of the              photoclick chemistry, the use of alkene reporters for directly
two isomers (cis and trans) with the two domains in Pin1               visualizing newly synthesized proteins with a spatiotemporal
(PPIase and WW) results in a complex reaction scheme. Our              control in live mammalian cells.
approach is to study the individual Pin1 domains and use
information thus obtained to elucidate the complete reaction           130
scheme. We are using NMR lineshape analysis, which is very
sensitive to chemical exchange in the biologically relevant            Construction of an improved optogenetic chloride sensor
µs-ms timescale. The strength of lineshape analysis lies in            using cell-free protein engineering techniques
its ability to handle complex reaction schemes involving               Joshua Grimley1, Weina Wang1, Li Li2, Lorena Beese1,
multiple species. Since the number of fitted parameters                George Augustine2, Homme Hellinga1
increases rapidly with the complexity of the reaction scheme,
                                                                        Biochemistry, Duke University, Nanaline Duke Bldg., 307
we are using ITC to independently measure the binding                  Research Dr., Durham, NC, 27710, 2Neurobiology, Duke
constants, thereby ensuring that the lineshape analysis                University, Bryan Research Bldg., 311 Research Dr., Durham,
provides meaningful results. Using this approach we are able           NC, 27710
to characterize the dynamics of the substrate binding loop in          Advanced optogenetic tools enable real-time visualization
the isolated WW domain. These loop dynamics have been                  of subcellular events at the molecular level. Clomeleon, a
implicated in the specificity of Pin1-WW domain towards                previously described CFP–YFP fusion, was serendipitously
phosphorylated substrates. The loop dynamics are sensed by             discovered as a probe for monitoring neuronal chloride.
most residues in the domain and appear to be governed by the           Chloride binds to YFP causing a ligand-dependent loss of
tautomeric state of the lone histidine sidechain in the domain.        fluorescence. The CFP provides a partner for fluorescence
This work is supported by the National Institutes of Health            resonance energy transfer, allowing ratiometric determination
(NIH R01-AG029385).                                                    of chloride concentration. However, the applicability of
                                                                       Clomeleon as a chloride sensor in vivo is limited by its low
                                                                       affinity for chloride (KD = 167 mM) relative to the intracellular
Imaging/Biosensors (129 – 137)                                         neuronal chloride concentration of 5 mM.
                                                                       To improve Clomeleon’s chloride-sensing ability and to
129                                                                    better understand how proteins coordinate halides, we carried
A bioorthogonal chemistry approach to visualizing                      out an enumerative mutagenic screen of seven residues in the
proteins in live mammalian cells                                       halide binding site of YFP. Single mutants were tested first,
Qing Lin, Wenjiao Song, Zhipeng Yu, Zhiyong Wang, Reyna                followed by combinations of double and triple mutants. We
Lim                                                                    used automated gene synthesis to generate 422 unique YFP
Chemistry, State University of New York at Buffalo, 679                open reading frames from 353 synthetic oligoneucleotides.
Natural Science Complex, Buffalo, NY, 14260                            Of these, 290 were expressed using a cell-free in vitro
                                                                       transcription-and-translation system and tested in a high-
The use of small organic groups, such as aldehyde, ketone,
                                                                       throughput fluorescence assay.
azide and alkyne, as bioorthogonal chemical reporters for
protein visualization in vivo has attracted intense interests          This procedure yielded a variant of YFP that was five
recently. Compared to genetically encoded fluorescent                  times more sensitive than the initial construct. Additional
proteins, chemical reporters are less likely to perturb the            mutations outside of the chloride binding site that have been


previously demonstrated to increase fluorescence and energy          the wild type) and is over 100 fold selective over dopamine
transfer were also incorporated. When combined with the              and norepinephrine. This protein displays serotonin induced
two binding site mutations, the new sensor was almost ten            T1 relaxation changes and is currently being investigated as
times more sensitive than Clomeleon. These results have              an fMRI sensor to monitor serotonin release in cell culture.
been confirmed with ex vivo imaging assays of primary                This study represents the first fMRI-based sensor approach for
murine neuronal cells. We undertook crystallographic studies         monitoring serotonin release from cells. This strategy should
of several YFP variants with four halides to understand              provide a general means of developing MRI contrast agents
the differential binding affinities covering three orders of         that respond to a variety of small molecules of biological
magnitude. In addition to two previously reported binding            importance.
sites, three new halide binding sites have been identified
near the chromophore. We are currently investigating halide          132
recognition in YFP and improving the sensor’s spectral
properties.                                                          A Nanoarray-based Immunobiosensor for the Detection
                                                                     of an Antibody Secreted by a B-Cell Hybridoma
This research was supported by a grant from the Duke                 Naveed Gulzar1, Donna Hohertz2, Christy Hui4, Rajan
Institute for Brain Sciences.                                        Nirwan4, Sean Romanuik3, Samantha Grist3, Reuven Gordon5,
                                                                     Bonnie Gray3, Alexandre Brolo4, Karen Kavanagh2, Jamie
131                                                                  Scott6
Directed Evolution of Protein-Based Neurotransmitter
                                                                      Dept. of Molecular Biology and Biochemistry, 2Dept.
Sensors for Functional MRI                                           of Physics, 3Microinstrumentation Laboratory, School
Eric Brustad1, Fay Bi1, Victor Lelyveld2, Alan Jasanoff2,            of Engineering Science, Simon Fraser University, 8888
Frances Arnold1                                                      University Drive, Burnaby, BC, V5A 1S6, Canada, 4Dept. of
  Chemical Engineering, California Institute of Technology,          Chemistry, 5Dept. of Electrical and Computer Engineering,
1200 E. California Blvd, MC 210-41, Pasadena, CA,                    University of Victoria, 3800 Finnerty Road, Victoria, BC, V8P
91125, 2Biological Engineering, Massachusetts Institute of           5C2, Canada, 6Dept. of Molecular Biology and Biochemistry,
Technology, MIT Building NW14, Rm. 2324, 150 Albany                  Simon Fraser University, 8888 University Drive, Burnaby,
St., Cambridge, MA, 02139                                            V5A 1S6, Canada
Functional magnetic resonance imaging (fMRI) of neural               The isolation of monoclonal (M) antibodies (Abs) from Ab-
markers would provide a powerful non-invasive approach               secreting cells (ASCs) is a laborious and time-consuming
to study signaling events that occur in the brain with high          process. Furthermore, it is difficult to detect and identify
spatial and temporal resolution. We have developed protein-          specific Abs of interest from cells whose frequency is relatively
based fMRI contrast agents using cytochrome P450-BM3                 low in a heterogenous cell population. Here, we describe
from Bacillus Megaterium for the selective visualization of          progress in the development of a platform for detecting the
neurotransmitters in vivo. We have previously demonstrated           binding to antigen (Ag) of Abs secreted by a single ASC. Our
that the isolated heme domain of P450-BM3 (BM3h) exhibits            team is using surface plasmon resonance to detect binding of
native MRI contrast capabilities due to the interaction of a         MAbs to Ag immobilized on an array of gold nanoholes, and
water molecule with the spin-active heme ligand of BM3h.             from there, to detect Ab secreted from hybridoma and plasma
Furthermore, the addition of a cognate fatty acid substrate          cells. Slides were fabricated bearing arrays of nanoholes in
to BM3h yields a dose dependent decrease in MRI signal,              a gold film and coated with complexes of streptavidin (SA)
which suggests that this protein would allow the selective           and a biotinylated (bio) peptide, mimicking large protein
visualization of substrate binding using fMRI. We have               Ags, specific for one of two MAbs: 2F5 and 17/9. MAb
developed an absorbance based P450 ligand-binding                    binding was measured by recording white light transmission
assay to allow the high-throughput screening of libraries            spectra over a wavelength range of 300 –1000 nm. Using a
of BM3h variants. In this way, directed protein evolution            microfluidic channel, 2F5 MAb (5 nM to 83 nM) binding to
can be applied to alter the substrate specificity of the BM3h        immobilized bio-2F5 peptide on a nanoarray surface resulted
active site providing tailored MRI contrast agents using a           in a change in peak wavelength intensity from 1.0 – 17.5
single protein scaffold. We have used this high-throughput           nm. Measured in air, 17/9 MAb (10 nM to 166 nM) binding
screening approach in combination with active site-directed          to bio-HA resulted in a shift of 1.0 – 3.7 nm. Binding of
saturation mutagenesis and error prone PCR to select for             Ab secreted by ~100 hybridoma cells to Ag on the nanoarray
BM3h based sensors that show high affinity and specificity           sensor produced a shift of ~12 nm; Ab secreted by ~10 cells
for the monoamine neurotransmitters serotonin, dopamine              could not be detected. We have made significant progress
and norepinephrine. One variant (BM3h-2G9) possesses                 towards the goal of identifying single ASCs whose Abs
only three mutations from the wild type BM3h, yet binds              bind a targeted Ag, and the relative affinity of that Ab for
serotonin with 3.5 µM affinity (>400 fold improvement over           Ag. If successful, this technology should allow rapid, real-
                                                                     time identification of cells secreting Ag-specific Abs and the


cloning of expressed Ab genes from live cells. This work               Fluor 680 and Cy5.5 dyes. These dyes are well separated
was supported by NSERC Strategic grant (365230).                       from red fluorescent molecules such as Phycoerythrin,
                                                                       DyLight 594 and DyLight 649 making it suitable for
133                                                                    multiplexing applications. The new “Brighter” DyLight
                                                                       680B dye exhibits a property that has not been reported for
Synthesis of targeted fluorescent probes for super-                    any other dye. The “Brighter” free dye is similar in brightness
resolution cellular imaging of protein function                        to Alexa® Fluor 680 and Cy5.5 however upon conjugation
Na Fu, Yijia Xiong, Curt Boschek, Uljana Mayer, Thomas                 to protein it exhibits enhancement in fluorescence intensity.
Squier                                                                 This allows conjugates made with DyLight 680B to enhance
Cell Biology & Biochemistry, Pacific Northwest National                signal intensity and sensitivity by 3-10 fold. Here we have
Laboratory, 902 Battelle Boulevard, Richland, WA, 99352                demonstrated the signal intensity and sensitivity obtained
With the goal of identifying linkages between cellular location        with DyLight 680B conjugates in fluorescence plate based
and coupled structural changes linked to protein function,             assays and immunofluorescence applications.
we have synthesized a new family of cell-permeable small
organic photoswitchable fluorophores that target and tightly           135
bind through a tetracoordinate linkage through a biarsenical
AsCy3 scaffold to a unique tagging sequences engineered into           Nanobody-based biosensor detecting metastasizing breast
protein backbones (1). Attachment of a proximal dye (e.g.,             cancer cells
Cy5) within the AsCy3 scaffold acts to form an optical switch          Marek Malecki MD PhD1, Raf Malecki2
(AsCy3-Cy5), similar to that previously used to obtain super-
                                                                        Western University, 309 E. Second Street, Pomona, CA,
resolution (subdiffraction) imaging following the labeling             91766-1854, 2NVI, 252 Montana Street, San Francisco, CA,
of antibodies with Cy3 and Cy5 dyes (2, 3). However, in                94112
contrast to antibody based probes, AsCy3-Cy5 retains the               Nearly 200,000 women will be diagnosed with breast cancer
small size and cell permeability necessary for both live cell          and 40,910 of them will die in the U.S.A. in 2010 according
imaging applications and protein functional measurements.              to the National Cancer Institute. Therapy of primary tumor
Currently, the photophysics and mechanisms of action that              is often successful, but metastases to the tissues, which
promote light-dependent switching necessary for STORM                  are distant from the primary tumor often escape diagnostic
imaging are under investigation. It is expected that AsCy3-            processes and locally administered therapy of surgery or
Cy5 will provide a robust new capability to track the cellular         radiotherapy. Cancer metastasizes via blood and/or lymph.
location or the dynamics of single proteins in cells.                  Therefore, detection of these metastasizing cells is of critical
1. Cao, H.; Xiong, Y.; Wang, T.; Squier, T. C.; Mayer, M. U.;          importance to eliminate them.
   J. Am. Chem. Soc., 2007, 8672                                       Recently, we genetically engineered single chain variable
2. Bates, M.; Blosser, T.R.; Zhuang, X. Phys. Rev. Lett. 2005,         fragments targeting specific domains of molecules present on
   94, 108101                                                          the breast and ovarian cancer cells. Further, we manufactured
                                                                       microarrays capable for selective anchoring of the cancer
3. Conley, R. N.; Biteen, S. J.; Moerner, E. W.; J. Phys. Chem.        cells expressing these molecules (e.g., epidermal growth
   B., 2008, 11878                                                     factors receptor 1-4 and their mutations). By applying the
                                                                       cancer patients’ blood to these microarrays, we were able
134                                                                    to anchor the cancer cells, apparently present in the tested
New DyLight 680 “Brighter” Far-Red Dye for Labeling                    blood, to the spots on microarrays. Using antibodies, we were
and Detection                                                          able to further label these cells’ cancer specific markers (e.g.,
Boguslawa Dworecki1, Marie Nlend1, Surbhi Desahi1, Janaki              intermediate filaments). The anchored cells were imaged with
Narahari1, Steve Shiflett2                                             the laser scanning confocal fluorescence on the microchips.
  Research & Development, 2Marketing, Thermo Fisher                    This technology opens new avenues for detecting and
Scientific, 3747 N Meridian Road, Rockford, IL, 61101                  diagnosis of metastasizing cancer and plan new therapeutic
Fluorescent dyes exhibiting Far-Red and Near Infra-Red
excitation are becoming powerful tools in life science research        Our work is conducted with the IRB Protocol, 09/IRB/035.
because they exhibit high sensitivity, low background, broad
linear dynamic range, minimal autofluorescence and light
scattering. Thermo Scientific is introducing a “Brighter”
DyLight 680B dye that is excited at 679 (± 4nm) and exhibits
fluorescence emission at 698 (± 4 nm). The excitation and
emission wavelengths for the new “Brighter” DyLight 680B
are similar to our existing photostable DyLight 680, Alexa®


136                                                                     design and construction of a comprehensive series of sensors
                                                                        for the profiling of 10 caspase family members in in vitro
A three-hybrid split-luciferase sensor for profiling protein            translation systems as well as human cell extracts. We found
kinases                                                                 that a reticulocyte lysate system possess significant caspase
Benjamin Jester, Alicia Gaj, Carolyn Shomin, Kurt Cox,                  dependent activity, whereas a wheat germ lysate system
Ping Wang, Indraneel Ghosh                                              provides a convenient expression system for establishing
Department of Chemistry and Biochemistry, University of                 the substrate specificity profile of caspases-1 through 10.
Arizona, 1306 E University Blvd, Tucson, AZ, 85721                      Finally, we demonstrate that the designed biosensors provide
The study of protein kinases and their inhibitors is important          a convenient method for understanding caspase activation in
for the elucidation of their cellular function as well as in            mammalian cell extracts.
the treatment of disease. The human genome encodes over
500 highly conserved protein kinases and the overwhelming
majority of known kinase inhibitors are promiscuous. Most
existing laboratory methods require radio-labeled substrates
and purified kinases and are not routinely amenable for
generating inhibitor selectivity profiles against a large set of
kinases. To this end we have developed a general, three-hybrid
approach for the rapid screening of small molecule-protein
interactions and applied it to protein kinases. Fragmented
halves of firefly luciferase were fused to either a protein
kinase domain or one member of a coiled-coil interacting
pair. The complementary coiled-coil partner was conjugated
to an active-site directed small molecule, and inclusion of             1 Shekhawat, S.S., Poter, J.R., Prasad, A., and Ghosh., I
the peptide-ligand conjugate with the split-luciferase sensor             (2009) An auto-inhibited coiled-coil design strategy for
resulted in generation of active reassembled luciferase. The              split-protein protease sensors, J. Am. Chem. Soc. 131,
kinase split-luciferase biosensor can be expressed in lysate              15284-15290
and assayed directly, obviating the need for purification and
allowing for rapid kinase inhibitor profiling. Moreover, this           This research was supported by grants from NSF (CHE-
approach also allows for determining the role of particular             0548264) and NIH (R01AI068414)
residues or domains in a kinase that are implicated in ligand
binding. To date we have demonstrated the potential of this
methodology with over 150 protein kinases from all arms of
                                                                        Intracellular Trafficking and
the human kinome and interrogated a library of 80 known                 Secretion (138 – 140)
small molecule kinase inhibitors.
                                                                        Polyubiquitin recognition by tandem ubiquitin binding
An Autoinhibited Coiled-Coil Design Strategy For Split-                 domains of Rabex-5
Protein Protease Sensors: Application To Caspases                       Sei Young Lee, Dong Hyuk Shin, Sangho Lee
Sujan Shekhawat, Jennifer Furman, Jason Porter, Akshay                  Department of Biological Sciences, Sungkyunkwan
Sriprasad, Indraneel Ghosh                                              University, 300 Cheoncheon-dong, Suwon, 440-746, Korea,
Chemistry and Biochemistry, University of Arizona, 1306                 Republic of (South)
University Blvd., Tucson, AZ, 85721                                     Polyubiquitination with various linkages among constituent
Caspases are intracellular cysteine proteases which have been           ubiquitin molecules serves as either degradatory or regulatory
implicated in apoptosis, cell proliferation, differentiation            signals. Linear polyubiquitination, by which ubiquitin
and inflammation. We have recently developed an auto-                   molecules are connected in head-to-tail fashion and the
inhibited coiled-coil design strategy for detecting protease            resulting chain is covalently attached to a target protein, is
activity using genetically encoded turn-on biosensors1.                 involved in the activation of NF-κB signaling. Such linear
Our split-protein sensor is auto-inhibited in the absence               polyubiquitin chain is known to be recognized by UBAN
of protease of interest; addition of protease, results in the           domain of NEMO. Despite of the emerging significance of
complementation of the split-protein fragments mediated                 linear polyubiquitination in vivo, the biochemical studies of
by coiled-coil dimerization. Three generations of protease              the domains recognizing the linear polyubiquitin chain lag
biosensors have been constructed using this strategy, with the          behind. Here we report that the N-terminal fragment of Rabex-5
final design providing a 1000-fold increase in bioluminescent           which contains tandem ubiquitin binding domains - A20-
signal upon the addition of a specific protease. We report the          type zinc finger domain (A20_ZF) and motif-interacting with


ubiquitin (MIU) - binds linear tetraubiquitin with the affinity           140
of 10 μM, which is comparable to that with monoubiquitin.
Site-directed mutagenesis studies suggest that both A20_ZF                A pH-regulated folding switch in Pseudomonas syringae
and MIU contribute to the linear tetraubiquitin binding with              effector protein AvrPto modulates its translocation via
similar affinities. We find that the N-terminal fragment of               the type III secretion system
Rabex-5 also binds K63-linked tetraubiquitin, but not K48-                Jolita Seckute1, Jennifer Dawson1, Brian Kvitko2,3, Alan
linked one. Previous structural studies have established                  Collmer2, Linda Nicholson1
that A20_ZF recognizes the non-canonical polar patch on
                                                                           Department of Molecular Biology and Genetics, Cornell
ubiquitin while MIU the canonical hydrophobic patch. Our                  University, 241 Biotechnology Building, Ithaca, NY, 14853,
results suggest that the recognition of linear polyubiquitin as
                                                                           Department of Plant Pathology and Plant-Microbe Biology,
well as K63-lined and K48-linked ones by Rabex-5 include                  Cornell University, 302 Plant Science Building, Ithaca, NY,
both canonical hydrophobic and non-canonical polar patches                14853, 3IDRC, Colorado State University, 3205 Rampart Rd,
on ubiquitin.                                                             Fort Collins, CO, 80526
                                                                          Bacteria have evolved secretion systems as direct conduits
139                                                                       into host cell cytoplasm for bacterial effector protein
                                                                          trafficking to further their symbiotic or pathogenic purposes,
Protease and Hemolysin Production by Urogenital Isolates                  while avoiding detection and host immune responses. Gram-
of E. faecalis                                                            negative bacterium Pseudomonas syringae uses type 3
Demetrius Coombs, Paula Porter, Lawrence W. Anderson                      secretion system that spans bacterial membranes and extends
Biology, Westminster College, 1840 South 1300 East, Salt                  with a needle-like protrusion to open up through the host
Lake City, UT, 84105                                                      cell membrane. There are structural constraints to passage
Enterococcus faecalis is a Gram positive coccus commonly                  through this conduit, where the inner channel diameter of
found amongst the normal flora inhabiting the intestinal tracts           ~2.8 nm prevents effector protein transport in the folded
of animals and is a leading cause of nosocomial infections in             form. The requirement for transient unfolding places unique
humans, particularly of the urinary tract. This investigation             restrictions on the stability thresholds for effector structure
examined the relative frequency and concentration of two                  and function, essential for bacterial pathogenicity. Effector
virulence factors, specifically a hemolysin and a (cytotoxin)             tertiary structure is typically needed for function in the host, as
protease, and the conditions affecting their regulation. We               has been shown for one of the most widely studied effectors,
hypothesize that altering the culture environment to simulate             AvrPto. AvrPto is an 18-kDa protein with helical core of low
the urogenital tract in vitro will have a profound effect on the          stability and disordered N- and C-termini, secreted by the
production of these virulence factors. Nine urogenital clinical           plant pathogen Pseudomonas syringae. We have previously
isolates were obtained and their identity confirmed using the             studied the unfolding behavior of this effector as a function
Remel RapID STR System. The microbes were grown in                        of pH in the range expected in the biological system, from
TSA medium with and without urine. The isolates were then                 acidic apoplast to neutral plant cell cytoplasm. We determined
tested for proteolytic activity on milk agar, and for hemolysin           the presence of a single amino acid folding switch governed
activity on Sheep’s Blood Agar under aerobic and anaerobic                by histidine 87 (H87) in AvrPto that acts in this pH range.
conditions. Three strains produced proteolytic activity simply            Here we present the effect of disabling this switch by a
by being grown in TSA. Two additional strains exhibited                   point mutation that replaces H87 with tyrosine (H87Y). We
proteolytic activity, but only after growth in medium containing          studied the translocation efficiency of WT and H87Y mutant
urine. Significant increases in urine concentration did not               in planta at different temperatures, and compared the results
demonstrate a corresponding increase in protease production               with in vitro unfolded population data at acidic pH values.
in these two strains. One strain exhibited hemolytic activity             We conclude that the intracellular bacterial pH is expected
but only when grown under anaerobic conditions. No strains                to be below pH 6 for efficient AvrPto translocation, and we
demonstrated both hemolytic and proteolytic activity under                present evidence that effector stability is a significant factor
our growth conditions. Proteolytic activity was confirmed                 in the translocation process Source of research support: NSF
and measured using quantitative fluorometry and a Pierce                  Grant MCB-0641582.
Fluorescent Protease Assay Kit after ammonium sulfate
precipitation. Growth in urine increased proteolytic activity by
5-8 times. These results suggest that proteolytic and hemolytic
activity of E. faecalis is associated with at least some clinical
isolates. The experiments also demonstrate the up-regulation
of protease gene expression after growth in broth containing
urine. The observed increase in protease activity in the
presence of urine implicates this protease as having a possible
role in clinical urinary infections and disease.


Membrane Proteins (141 – 149)                                          (D-CF) or post-translational (P-CF) solubilization strategies
                                                                       in supplemented detergent micelles; by (II) expression in
                                                                       presence of specific ligands; by (III) providing defined
                                                                       lipid environments like liposomes or nanodiscs and by (IV)
NMR Studies of the Structure, Dynamics, and Interactions               modification of posttranslational downstream processes.
of CXCR1 and IL-8                                                      Exemplified with the human endothelin A (ETA) and
Fabio Casagrande, Leah Cho, Lauren Albrecht, Mignon                    endothelin B (ETB) receptors representing the central
Chu, Sang Ho Park, Stanley Opella                                      network involved in blood pressure regulation, heart diseases
Chemistry and Biochemistry, UCSD, 9500 Gilman Drive, La                and pulmonary dysfunction, we present strategies for
Jolla, CA, 92093-0307                                                  expression protocol design in individual CF systems. Our
The small chemokine ligand interleukin-8 (IL-8) induces                optimized production processes yield in the synthesis of
activation and cell migration of polymorphonuclear neutrophils         several mgs of GPCRs per single ml of CF reaction in less
by binding to the G-protein coupled receptor CXCR1. Defects            than 24 hours. The GPCR sample quality was characterized
in signal transduction result in inflammatory disorders such           by complementary approaches including homogeneity,
as allergies, rheumatoid arthritis, and contribute to cancer           protein stability, formation of homo- and hetero-oligomeric
growth and metastasis. New structural and functional insights          complexes and ligand binding kinetics. We demonstrate
would provide an opportunity to improve the prospects for              that CF expression in the P-CF as well as in the D-CF mode
new drugs applicable to a broad range of human diseases.               can result in homogenous and highly stable GPCR samples
The presented studies focus on the dynamics of CXCR1 and               containing up to 70 % ligand binding competent receptors.
its interaction with IL-8. Our approach using a combination
of solution NMR and solid-state NMR spectroscopy offers                143
the possibility to investigate CXCR1 in micelles, bicelles,
and planar phospholipid bilayers. A range of full-length and           An In-Membrane Hydropathy Scale Developed with
truncated CXCR1 constructs and IL-8 proteins have been                 CFTR Constructs
expressed, isotopically labeled and purified to homogeneity.           Cory Mulvihill1,2, Charles Deber1,2
The individual proteins and their complexes were reconstituted
                                                                        Molecular Structure & Function, Hospital for Sick Children,
in the presence of lipids. Progress towards determining the            555 University Ave., Toronto, ON, M5G 1X8, Canada,
three-dimensional structure of CXCR1 along and complexed
                                                                        Department of Biochemistry, University of Toronto, 1 King’s
with IL-8 will be presented.                                           College Circle, Toronto, ON, M5S 1A8, Canada
                                                                       Diseases such as cystic fibrosis (CF) and retinitis pigmentosa
142                                                                    result from genetic defects that lead to misfolding of
                                                                       membrane proteins1,2. While there is extensive knowledge of
Preparative scale cell-free production of G-protein                    the misfolding of soluble protein domains, our understanding
coupled receptors in high quality                                      of the hierarchy of forces that guide folding - and misfolding
Friederike Junge, Christian Roos, Volker Doetsch, Frank                - of integral membrane domains remains limited. Here we
Bernhard                                                               use helix-loop-helix (‘helical hairpin’) constructs of the
Biophysical Chemistry, Goethe University Frankfurt/Main,               cystic fibrosis transmembrane conductance regulator (CFTR)
Max-von-Laue Straße 9, Frankfurt/Main, Germany                         to model the structural consequences of lesions in membrane
G-protein coupled receptors (GPCRs) are key players in                 spanning domains. Such helical hairpins, composed of
signal perception and transduction. Their essential roles              CFTR transmembrane (TM) helices 3 and 4, have previously
in pathophysiological processes make them to one of the                been characterized in our laboratory and shown to be a
most important drug targets in pharmacological research.               relevant model3. To further elucidate residue-dependent
However, structural and functional studies are still hampered          intramembrane folding and stability, TM3/4 hairpins with
by tremendous difficulties in producing suitable amounts of            each of the 20 amino acids at position 232 were individually
protein in conventional cell based expression systems.                 expressed in E. coli, isolated, and characterized. Retention
Cell-free (CF) expression has emerged as powerful tool to              times on reverse phase (RP) HPLC, in combination with
overcome major restrictions of classical in vivo membrane              circular dichroism spectroscopy, were used to determine the
protein production systems such as efficient targeting,                biophysical/structural effects of position 232 substitutions.
translocation or even toxicity to the cellular hosts. The              In addition, since mobility on SDS-PAGE has recently been
open nature and high versatility of CF expression offers               shown by our laboratory to be primarily a measure of detergent
a variety of completely new options to rationally design               binding, as well as a function of hydropathy and SDS-
membrane protein expression environments. We have                      resistant tertiary contacts between the two TM helices4, we
modulated yield and quality of CF expressed GPCRs by (I)               have analyzed the SDS-PAGE mobilities of the CFTR TM3/4
different expression modes implementing co-translational               V232X hairpin library as a function of residue character. The


combined results are used to produce a novel “in-membrane”             we hope to demonstrate that SMALP will offer a universal
hydropathy scale for the 20 amino acids. Comparisons with              method of producing pure membrane protein solubilised in
the in vivo scale for membrane insertion reported by Hessa et          their native membranes.
al.5 indicate strong correlation between the two.
1. Wallin, E. & von Heijne, G. Protein Sci. 7, 1029 (1998).            145
2. Kuznetsov, G. & Nigam, S. K. N. Engl. J. Med. 339, 1688             Binding pocket analysis of seven helical transmembrane
   (1998).                                                             proteins: Is sequence identity alone suitable for modeling
3. Therien, A. G., Grant, F. E. M. & Deber, C. M. Nat. Struc.          GPCR as drug targets ?
   Biol. 8, 597 (2001).                                                Vagmita Pabuwal1, Zhijun Li2,3
                                                                        Chemistry & Biochemsitry, 2Dept. of Bioinformatics and
4. Rath, A., et al. Proc. Natl. Acad. Sci. U.S.A. 106, 1760
                                                                       Computer Science, University of the Sciences in Philadelphia,
                                                                       600 S 43rd Street, Philadelphia, PA, 19104, 3Institute for
5. Hessa, T. et al. Nature 433, 377 (2005).                            Translational Medicine and Therapeutics, University of
                                                                       Pennsylvania, 3400 Spruce Street, Philadelphia, PA, 19104
144                                                                    Transmembrane proteins are responsible for various
Over-expression and biophysical characterisation of                    physiological and pathological mechanisms in human body.
membrane proteins solubilised in a styrene maleic acid                 They account for ~30% of human genome which makes it
copolymer                                                              important to understand their structure-function relationships.
yupin lin1, Raul Pacheco-Gomez1, Ying-jia Yu1, Hsing-wen               Seven helical transmembrane proteins including the G-protein
Huang1, Mohammed Jamshad1, Timothy Knowles2, Corinne                   coupled receptors (GPCR), accounts for ~26% of current
Smith3, Michael Overduin2, Timothy Dafforn1                            drug targets and have proven to be important and lucrative
  School of Biosciences, 2School of Cancer Sciences,                   pharmaceutical targets. In order to gain novel insight into its
University of Birmingham, Edgbaston, Birmingham, B15                   structure-function relationships, we studied high-resolution
2TT, United Kingdom, 3Department of Biological Sciences,               structure of transmembrane proteins from a perspective of
University of Warwick, University of Warwick, Coventry,                binding site analysis. Various computational methods have
CV4 7AL, United Kingdom                                                been introduced so far for the binding site comparison of
                                                                       soluble proteins. In our work we compare the binding sites
Membrane proteins are the gateways into cells and therefore
                                                                       of these seven helical membrane proteins using Cavbase, a
a critical part of pharmaceutical investigations. In fact,
                                                                       software taking into account the physicochemical properties
membrane proteins have been shown to be the target of over
                                                                       and shape of the side chain residues at the binding site for the
50% of all medicinal drugs. The development of drugs that
                                                                       comparison of any two binding sites. Our study suggests that
act on membrane proteins, however, has been limited by the
                                                                       structure with high sequence identity may have low structural
lack of high resolution data on their structures and function.
                                                                       similarity around binding site although these are suppose to
We have developed a novel styrene maleic acid (SMA)/
                                                                       be conserved structurally. This implies that we should not
lipid assembly termed SMALP that preserves membrane
                                                                       rely solely on sequence identity for selecting templates for
proteins structural integrity, keeping them monomeric and
                                                                       homology modeling of GPCRs.
active within a lipid-rich environment. SMALP has been
successfully applied to the investigation of α-helical bundle,
β-barrel transmembrane proteins and human G-protein                    146
coupled receptors. Together with standard purification                 Transmembrane protein segments: post-translational
protocols, pure membrane proteins were obtained without                rearrangements and integration into the membrane of
any detergents added. Our recent work showed SMALP                     α-helices of low hydrophobicity
has been optimized for solubilisation of ZipA, a membrane-             Linnea Hedin1, Karin Öjemalm1, Anni Kauko2, Aron
anchored protein which is an essential part of the machinery           Hennerdal1,3, Kristoffer Illergård1,3, IngMarie Nilsson1,
that underlies cell division in Escherichia coli. Analytical           Gunnar von Heijne1,3, Arne Elofsson1,3
studies for ZipA encapsulated in SMALP, using circular                 1
                                                                        Center for Biomembrane Research, Department of
dichroism (CD) and analytical ultracentrifugation (AUC),               Biochemistry and Biophysics, Stockholm University,
reveal pure SMALP solubilised ZipA to be natively folded,              Stockholm, SE-106 91, Sweden, 2Department of
with a size of 77 kDa. The interaction between ZipA and                Biochemistry and Pharmacy, Åbo Akademi University,
FtsZ, a homologue of eukaryotic tubulin, was also studied              Structural Bioinformatics Laboratory, Turku, 20520, Finland,
using right angled light scattering (RALS), linear dichroism           3
                                                                        Stockholm Bioinformatics Center, Stockholm University,
(LD) and a GTPase assay. These results indicate that ZipA              Stockholm, SE-106 91, Sweden
encapsulated in SMALP is functional and also provide useful
                                                                       In mammalian cells, most integral membrane proteins are
information about the ZipA-FtsZ interaction. Eventually
                                                                       initially inserted into the endoplasmic reticulum membrane


by the so-called Sec61 translocon. We have studied known                6xHis tag) and a spacer arm at the N-terminus, creating a
three-dimensional structures of multi-spanning integral                 novel affinity ligand to interface with various chromatography
membrane proteins. Using a hydrophobicity predictor based               media. The pharmacological properties of the affinity ligands
on a biological scale, we found (1) transmembrane helices               were tested (specific binding, activation and affinity). The UT
(TMHs) predicted to be insufficiently hydrophobic to be                 was stably expressed in mammalian cells (293-F or CHO-S)
recognized as such by the translocon. (2) TMHs of low                   and the utility of the ligands were investigated during
hydrophobicity having overlapping segments predicted                    purification of UT.
to be integrated into the membrane. Using recombinantly                 We found that the affinity ligands specifically bound and
introduced sites of N-linked glycosylation and in vitro                 activated the UT to varying degrees. Pre-binding of affinity
expression in the presence of microsomes, we have studied               ligands to the UT (before exposure to detergent) facilitated
the insertion efficiency of a number of these segments. Of 16           specific capture of UT by stabilizing the receptor structure
marginally hydrophobic TMHs, most did not insert efficiently            during detergent solubilization (with n-Dodecyl β-D-
into the endoplasmic reticulum membrane by themselves. The              maltoside). Captured UT complexes allowed co-capture of
presence of flanking loops and nearest-neighbor TMHs are                Gαq/11 protein and were resistant to dissociation at elevated
sufficient to ensure the insertion of all but two of the TMHs,          temperatures. This suggests that purified UT is able to adopt an
indicating that post-insertional rearrangements are involved            active conformation and is relatively thermo-stable, making
in the folding of these latter proteins. We also decided the            it an ideal candidate for future structural and biophysical
optimal placement of individual transmembrane helices in the            studies.
Pyrococcus horikoshii GltPh glutamate transporter homolog
in the membrane. The results are in close agreement with                This work demonstrates the value of ligand-based support of
the theoretical predictions based on hydrophobicity, but do             protein structure as a purification tool for GPCRs, resulting in
not, in general, match the known three-dimensional structure.           the first successful purification of the UT.
We conclude that for a number of α-helical proteins, folding
and integration into the membrane require co-translational              148
inter-molecular interactions and could also involve post-               Identification and Purification of Heliobacterium
translational rearrangments in respect to the plane of the              modesticaldum NADH:menaquinone Oxidoreductase
membrane.                                                               Hai Yue1, Robert Blankenship2
                                                                         Chemistry, Washington University in St.Louis, One
147                                                                     Brookings Drive, Washington University in St. Louis, Campus
Expression and purification of active state Urotensin-II                box 1134, Saint Louis, MO, 63130, 2Biology and Chemistry,
receptor with affinity ligands                                          Washington University in St.Louis, One Brookings Drive,
Ann Du1, Leonard Pattenden2, Mibel Aguilar1, Walter                     Washington University in St. Louis, Campus Box 1134, Saint
Thomas3                                                                 Louis, MO, 63130
  Biochemistry and Molecular Biology, Monash University,                Heliobacteria (Hb) are unique anaerobic anoxygenic
Wellington Rd, Clayton, 3800, Australia, 2Health Innovations            photoheterotrophs. In terms of carbon metabolism, Hb are
Research Institute, RMIT University, Plenty Rd, Bundoora,               obligately heterotrophic. Phylogenetically, Hb are the only
3083, Australia, 3The School of Biomedical Sciences, The                phototrophic organisms that group within the bacterial
University of Queensland, Sir Fred Schonell Dr, St Lucia,               phylum Firmicutes, most of which are Gram positive.
4072, Australia                                                         Finally, Hb are unique among all phototrophs in that they
A limitation for structural and biophysical analysis of G               produce endospores. Hb also contain the simplest known
protein-coupled receptors (GPCRs) is the inherent challenge             photosystem, with a homodimeric core reaction center (RC)
of purifying and stabilizing these receptors in an active               complex and no peripheral antenna. Finally, the heliobacterial
(agonist-bound) conformation. Peptide ligands, such as the              cytochrome bc complex appears to be a hybrid between
vasoactive, cyclic hormone Urotensin-II (U-II) are ideal                the bc1 and b6f complexes. With all of these remarkable
candidates for ligand-based affinity purification due to their          characteristics, however, our knowledge is limited in terms
high affinity and tight (pseudo-irreversible) binding. The              of the structure and function about almost every component
U-II receptor (UT), a receptor resistant to desensitization,            of the Hb photosystem. Based on previous biochemical and
displays an unusual propensity to remain active upon ligand             genomic research, we think that the NADH:menaquinone
binding. Our work aims to exploit these properties to express           oxidoreductase (NDH-1) complex may have an important
and purify UT at scales suitable for structural and biophysical         function in the photosynthetic electron transfer chain. To
analysis.                                                               characterize NDH-1 in Hb, we have utilized transcriptomics
                                                                        and proteomics methodologies, including Real Time
The U-II ligand was modified with affinity groups (biotin,              PCR, blue-native and SDS-PAGE, mass spectrometry and
desthiobiotin, Strep-tag II with or without a bifunctional              enzymatic activity assays. Based on preliminary results, we


think that NDH-1 is expressed with a fairly low level, yet              detergents as well as the biophysical characteristics of pore
is detectable within the membrane in terms of its NADH                  and channel formation.
dehydrogenase activity. After having identified its existence,
we are now trying to develop and optimize a protocol to purify
this huge complex and further analyze it with enzymatic                 New and Developing Methods
and biochemical techniques. At the same time, we will use
spectroscopic and electrochemical methodologies to further
                                                                        (150 –158)
broaden our understanding of the heliobacteria. This research
is supported by the Exobiology Program of NASA.
                                                                        Modeling of Protein Complexes Using Filamentary
149                                                                     Density Distributions
                                                                        Andrew Hausrath1, Alain Goriely3, Tsu-shuen Tsao2
Recapitulating the Native Local Environment of                          1
                                                                          Chemistry and Biochemistry, Program in Applied
Transmembrane Segments with Detergent Micelles                          Mathematics, 2Chemistry and Biochemistry, University
David Tulumello1,2, Charles Deber1,2                                    of Arizona, 1041 E Lowell, Tucson, AZ, 85721, 3Oxford
  Molecular Structure and Function, Hospital for Sick                   Centre for Collaborative Applied Mathematics, University
Children, 555 University Ave, Toronto, M5G 1X8, Canada,                 of Oxford, Mathematical Institute, 24-29 St Giles’, Oxford,
  Biochemistry, University of Toronto, 1 King’s College                 OX1 3LB, United Kingdom
Circle, Toronto, ON, M5S 1A8, Canada
                                                                        We describe a method for the representation of proteins
An inherent dilemma in the study of the structural biology              and their higher-order assemblies as geometrically defined
of membrane proteins is that the generation of high-                    density distributions based on the theory of space curves.
resolution structures (through x-ray crystallography and                Diverse types of data may be simulated from these models.
solution NMR) has overwhelmingly relied upon identifying                Here we discuss the basic theory for constructing these
detergents in which membrane proteins must be solubilized               filamentary distributions and the application of such models
without denaturation into a non-biological state. We have               to electron microscopy data. An advantage of the filamentary
recently observed that within transmembrane (TM) domains,               representation is that these density models may be defined
individual helices can be classified, based upon local                  at different resolutions, and therefore these models may
environment, into three categories; (i) exposure to lipid;              be constructed with an information content matching that
(ii) burial through helix-helix interactions; and (iii) aqueous         available from experiment. As a test case, we apply our
exposure through pore or channel formation, and that these              approach to construct models of the trimeric, hexameric, and
three states may be recapitulated using model TM segments               octadecameric assembly states of adiponectin.
within a detergent micelle environment [Tulumello & Deber,
Biochemistry 48, 12096-12103 (2009)]. To extend these
results to natural TM sequences, we have synthesized a
series of single TM segments corresponding to those found               Development of a Multiplex ELISA using Quantum Dots
in several membrane proteins for which a high-resolution                and Microarray Technology
structure has been determined. Peptides synthesized include             Sarah Schenk, Kerrm Yau, Todd Glancy
TM helix IV (ILAVFWISLYIFVNLTSVLYLGGLAL)                                Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN,
from vSGLT (Sodium Galactose Transporter), and TM                       46268
helices III (YLLWIITGMLVMFAPFFIFIFG) and V                              As the numbers of traits being combined into breeding stacks
(ARMFGCVGWALCASIVGIMF) from LacY (Lactose                               continues to increase, conventional methods of monitoring
Permease Transporter). Here we report the detergent solvation           and quantifying protein expression of these genes by detecting
and biophysical analysis of these segments through SDS-                 a single protein in one Enzyme-Linked Immunosorbent Assay
PAGE migration, and fluorescence and circular dichroism                 (ELISA) is becoming more problematic. As more genes are
spectroscopy. Results demonstrate that TM segments from                 placed in a single product, screening for protein expression is
the categories indicated above display differences in detergent         becoming more labor and resource intensive. The objective
solvation, and in secondary and quaternary structure. While             of this project was to develop a multiplex immunoassay
vSGLT helix IV and LacY helix V are largely alpha-helical,              to detect multiple proteins of interest simultaneously. A
we found that LacY TM helix III - a segment which lines an              hypothetical combination of proteins was used for this
aqueous exposed cavity - does not adopt regular secondary               study. Two methods for developing this assay were tested.
structure in common detergents tested. Preliminary results              The first was to label each of the detection antibodies with
of parallel analyses with TM segments from AdiC (Arginine:              quantum dots, which are inorganic fluorophores consisting
Agmatine Antiporter), and BtuCD (Vitamin B12 Transporter)               of luminescent semiconductor nanocrystals that emit at
will also be discussed. This work should provide insights into          different wavelengths based on size. The second method
the sequence requirements for stable TM domain folding in


was to spot the capture antibodies for each trait in a specific         153
spot on the assay plate using microarray technology. The
horseradish peroxidase (HRP) conjugated antibodies could                Coupling Somatic Hypermutation with Mammalian Cell
then bind specifically to their respective trait in a specific          Surface Display: A Novel Approach to the Discovery and
location in the well and be captured by a camera through                Optimization of Therapeutic Antibodies
chemiluminescence. Both the quantum dot and microarray                  Rachelle Toobian, Tamlyn Neben, Heather Jones, Jennifer
printed plates were able to simultaneously detect two of the            Dalton, Irina Krapf, Geoffery Tomlinson, John Macomber,
three proteins in maize matrix. The data indicate that through          Andy Chen, Laurence Altobell III, Trevin Holland, Ryan
further optimization, more traits can be added to this assay to         O’Hanlon, Betty Wu, Robert Horlick, David King, Peter
reduce number of assays to be performed, as well as reduce              Bowers
time and cost for protein analysis needed for commercial                AnaptysBio, Inc., 10835 Road to the Cure, Suite 100, San
product campaigns.                                                      Diego, CA, 92121
                                                                        Somatic hypermutation (SHM) is the process utilized in vivo
152                                                                     to evolve high affinity antibodies from germline sequences.
                                                                        SHM is initiated by activation-induced cytidine deaminase
Thermodynamic analysis of self assembly in coiled-coil                  (AID), a highly regulated B cell protein which acts directly on
biomaterials.                                                           specific DNA motifs in the antibody variable region. We have
Bashkim Kokona, Betty Tsang, Heidi Bretscher, Robert                    developed an in vitro antibody discovery and optimization
Fairman                                                                 platform by coupling the replication mutagenesis activity
Biology, Haverford College, 370 Lancaster Ave, Haverford,               of AID with mammalian cell surface display. Desired
PA, 19041                                                               antibody properties (such as higher affinity, cross-reactivity,
The role of the local interactions in the hydrophobic core              expression and stability) are evolved by co-expressing AID
of alpha-helical coiled coils is extensively studied in well-           with antibody heavy and light chains in HEK293 cells and
defined oligomers. Our laboratory designed a peptide which              selected using high-throughput FACS-based screening
is able to self-assemble onto large polymers as a result of a           methodology. We have demonstrated the ability to identify
phase shift of 200° of the C-terminal hydrophobic surface,              novel fully human antibodies from combined heavy and light
relative to the N-terminal hydrophobic surface. A family                chain libraries, and also achieved >1,000-fold improvement
of peptides was designed with the aim to better understand              in antibody binding after iterative selection in the presence of
the role of hydrophobicity at a buried a heptad position on             AID. Affinity of candidate antibodies was measured using
the stability, length and heterogeneity of the polymers. The            ELISA, Biacore T100 and A100, and KinExA 3000 assays.
degree of polymerization of these peptides is characterized             Sensitivity and robustness of the platform was verified using
using analytical ultracentrifugation, dynamic light scattering,         a series of control antibodies with a wide range of affinities
and atomic force microscopy. The dependence of self-                    that were subsequently recovered. This novel approach is
assembly on peptide concentration was studied using circular            currently being applied to the discovery and optimization of
dichroism. Utilizing a single step nucleation-elongation                antibodies to a variety of therapeutic targets.
model, we are able to quantitatively explain our results. The
stability of the polymers depends strongly on salt, as expected         154
for a process dominated by hydrophobic interactions; we
study the effects of salt on the thermodynamics of the self-            Tuning the stability of green fluorescence protein
assembly process. The majority of the polymerizing studies              promotes mutational robustness for non-canonical amino
are carried out with a hydrophobic core containing isoleucine           acids incorporation
at the a heptad positions and leucine at the d heptad positions         Nagasundarapandian        Soundrarajan,       Govindan
but we also characterize several mutants, containing either             Raghunathan, Sun-Gu Lee
serine, alanine, or asparagine at two of the four a heptad              Dept of Chemical Engineering, Biomolecular Engineering
positions. We will report on the effects of these mutations on          Lab, Pusan National University, 30, jangjeong-don,
nucleation and propagation parameters using this single-step            Geumjeong-gu, Busan, 609-735, Korea, Republic of (South)
polymer theory.                                                         In nature the ability of the protein to maintain the native fold
                                                                        and stability to perform biological activity is determined by
                                                                        the intrinsic property of the protein sequence itself. Recently,
                                                                        intensive researches have been carried out to insert novel
                                                                        physical and chemical properties into protein by incorporation
                                                                        of non-canonical amino acids (NCAA). However, in many
                                                                        cases the incorporation of NCAA into the protein often
                                                                        results in the loss of protein function by misfolding or
                                                                        aggregation. Here, we show that the enhancement of protein


stability permits us to avoid such misfolding problems. Since           gold cluster labeling on proteins and the labels as spatial
the incorporation of NCAA is technically mutagenesis, we                probes.
reasoned that the mutational robustness due to extra stability          Earlier in our research, we showed how to reconstruct gold
of protein may also make protein tolerant to the mutagenesis            cluster labeled protein structures and how to extract 1D
by NCAA. We have demonstrated that the stable green                     structural information (inter-body distances). Simulated
fluorescence protein (GFP) variants constructed based on                scattering curves of templates were compared to those of
superfolder GFP exhibited higher tolerance against NCAA                 trial structures, which were generated by rigid body searches
incorporation compared to wild type GFP. We compared the                for a fixed protein with mobile gold cluster(s). The gold
GFP variants and wild type by selective pressure incorporation          cluster labeled protein structures were reconstructed in two
methodology to incorporate various Met analogues such as                steps. First, inter-body distances were estimated from fine-
homopropargylglycine, azidohomoalaline, ethionine, and                  grid rigid body modeling (RBM) without rotation. Secondly,
norleucine. The analogues were chosen because they had                  gold center positions were predicted from distance-corrected
profound effects on the alteration of various interactions in           coarse-grid RBM. Inter-body distances of each trial structure
the protein, which may provide us a precise model to test               were corrected by least square minimization during RBM. In
our hypothesis. Only the stable GFP variants were able                  the analysis, double-labeled data were combined with single-
to tolerate the new side chain mutations, but the wild type             labeled data using the best performing scoring function, which
GFP often ended up in misfolding or aggregation due to the              improves the prediction accuracy of gold center positions (1.4
destabilizing mutations caused by Met analogues. We also                – 8.9 Å error). We also investigated the effect of inter-body
examined the refolding kinetics of the incorporated GFP                 distance error and grid error to obtain the nearest neighboring
variants. We conclude that contribution of extra stability              trial structure as a top scoring solution; these trials indicate
of the protein promotes mutational robustness for NCAA                  that 0.2 Å of inter-body distance error and 0.5 Å of grid error
incorporation.                                                          are the maximum allowed errors. To improve computation
Acknowledgement: This study was financially supported by                speed, in the second step, we plan to use scattering profile
Brain Korea 21 Program.                                                 generation with dummy atomic scattering factors and evenly-
                                                                        distributed angular searches.
155                                                                     1 Mathew-Fenn, R. S., R. Das, and P. A. B. Harbury. 2008.
Gold Cluster Labeling as a New Tool to Study Protein                      Remeasuring the double helix. Science 322:446-449.
Structures Using Small Angle X-ray Scattering                           2 Mastroianni, A. J., D. A. Sivak, P. L. Geissler, and A. P.
Hyun Chul Lee1, Bruce Craig2, Chris Bailey-Kellogg3, Chinh                Alivisatos. 2009. Probing the Conformational Distributions
Le4, Alan Friedman1                                                       of Subpersistence Length DNA. Biophys. J. 97:1408-
  Department of Biological Sciences, Purdue University, 915               1417.
W. State Street, West Lafayette, IN, 47907, 2Department of
Statistics, Purdue University, 250 N. University Street, West           156
Lafayette, IN, 47907, 3Department of Computer Sciences,
Dartmouth College, Sudikoff Lab: HB 6211, Hanover, NH,                  An ultra high-efficient system for in vivo and in vitro
03755, 4Rosen Center for Advanced Computing, Purdue                     synthesis of tyrosine-sulfated proteins
University, 151 South Grant Street, West Lafayette, IN,                 Lu-Yi Lu, Bo-Han Chen, Jennifer Wu, Chen-Chu Wang, Tsu-
47907                                                                   Hsiang Lin, Ching-Chen Hsu, Chih-Chi Lin, Yuh-Shyong
A new methodology for using gold cluster labeling as                    Department of Biological Science and Technology, National
special probes in solution X-ray scattering is presented.               Chiao Tung University, R102, Chu-Ming Building, 75 Po-Ai
Gold cluster labeling provides strong scattering interferences          Street, Hsinchu, 300, Taiwan
between gold atoms and other atoms. These strong scattering
interferences emphasize the structural information of proteins.         Protein tyrosine sulfation, catalyzed by membrane-associated
The scattering interference between gold clusters gives the             tyrosylprotein sulfotransferase (TPST, EC, is a key
cluster-to-cluster distance, while the scattering interference          post-translational modification that mediates various critical
between a protein and a gold cluster gives the protein-to-              functions, such as HIV entry, inflammation, coagulation, and
cluster distance and the gold cluster orientation relative to           sterility. Although much research has been devoted to decipher
the protein.                                                            the significances of protein tyrosine sulfation, the acquirement
                                                                        of sulfated proteins for further studies is still a challenge.
Recently, the application of gold cluster labeling on double-           Enzymatic catalysis provides a direct, specific and easy
stranded DNA in small angle X-ray Scattering was studied by             method for preparation and identification of sulfated proteins
Mathew-Fenn et al. (1) and Mastroianni et al. (2) and showed            in spite of the source of TPST, acquired mainly from natural
the successful role of gold cluster labels as molecular rulers.         tissues or cell cultures, is the main obstacle. In this report, the
So far, no research has been reported on the application of             constant source of active and homogeneous TPST at large


quantity was first achieved through prokaryotic expression. A          158
continuous 3¡¦-phosphoadenosine-5¡¦-phosphosulfate (PAPS)
in situ regenerating system was developed to provide activated         Crystallizing Proteins by Synthetic Symmetrization
sulfate group for TPST catalyzed reaction. This methodology            Garry Forse, Nina Ram, Michael Sawaya, Todd Yeates,
rendered increase in catalytic efficiency of TPST to 10-100            Duilio Cascio
folds higher than those obtained by previous studies. Using            Chemistry and Biochemistry, UCLA, 611 Charles Young Dr.
similar strategy, sulfated proteins were produced in vivo              East, Los Angeles, CA, 90095
with extremely high efficiency through bacterial cultivation           Protein crystallization continues to be a major bottleneck
incorporated with suitable genes. Recently, we applied this            in X-ray crystallography. Previous studies suggest that
platform to produce sulfated proteins in tissue extract and            symmetric proteins, such as homodimers, might crystallize
further identify them via western blotting and MALDI-                  more readily than asymmetric, monomeric proteins. Proteins
TOF. This platform will be beneficial to not only synthesize           that are naturally monomeric can be made homodimeric
sulfated proteins but investigate the functions of protein             artificially. One approach is to introduce single cysteines
tyrosine sulfation.                                                    into the protein of interest and then form a disulfide bond
                                                                       between the two monomers. By introducing the single
157                                                                    cysteine at different sequence positions, this approach can be
                                                                       used to produce a variety of synthetically dimerized versions
Self assembly of TMAO at hydrophobic interfaces and its                of a protein, with each construct expected to exhibit its own
effect on protein adsorption: Insights from experiments                crystallization behavior. In earlier work (Banatao, et al.
and simulations                                                        2006. PNAS 103, 16230-5), we demonstrated the potential
Gaurav Anand, Sumanth Jamadagni, Shekhar Garde,                        utility of the approach using T4 lysozyme as a model system.
Georges Belfort                                                        Here we report the successful application of the method to a
Department of Chemical and Biological Engineering,                     protein that had resisted crystallization its natural monomeric
Rensselaer Polytechnic Institute, 110 Eighth Street, Troy,             form, and had therefore fallen out of a structural genomics
NY, 12180                                                              pipeline for structure determination. The crystal structure
We offer a novel process to render hydrophobic surfaces                was determined for T. maritima CelA, a protein with less than
resistant to relatively small proteins during adsorption.              20% sequence identity to proteins with structures available
This was accomplished by self-assembly of a well-known                 from the protein data bank. This is the first application of
natural osmolyte, trimethylamine oxide (TMAO), a small                 synthetic symmetrization to determine a novel protein
amphiphilic molecule, on a hydrophobic alkanethiol                     structure. The synthetic dimerization of the mutant D188C
surface. Measurments of lysozyme (LYS) adsorption on                   yielded well-diffracting crystals with molecules in a packing
several homogeneous substrates formed from functionalized              arrangement that would not have occurred with monomeric
alkanethiol self-assembled monolayers (SAMs) in the                    CelA. The new crystal form was solved by a MAD phasing
presence and absence of TMAO, and direct interaction                   experiment with seleno-Met derivative protein crystals.
energy between the protein and functionalized surfaces,                The results support the idea that synthetic symmetrization
demonstrate the protein-resistance properties of a non-                can be a useful approach for enlarging the search space for
covalently adsorbed self-assembled TMAO layer. Molecular               crystallizing monomeric proteins.
dynamics simulations clearly show that TMAO molecules
concentrate near the CH3-SAM surface and are preferentially
excluded from LYS. Interestingly, TMAO molecules adsorb                Protein Design and Protein
strongly on a hydrophobic CH3-SAM surface, but a trade-
off between hydrogen bonding with water, and hydrophobic
                                                                       Engineering (159 – 168)
interactions with the underlying substrate results in a non-
intuitive orientation of TMAO molecules at the interface.
Additionally, hydrophobic interactions, usually responsible            Antibody Fragment Engineering With Non-Canonical
for non-specific adsorption of proteins are weakly affected            Amino Acids
by TMAO. In addition to TMAO, other osmolytes (sucrose,                James Van Deventer, Tae Hyeon Yoo, David Tirrell
taurine and betaine) and a larger homologue of TMAO                    Chemistry and Chemical Engineering, California Institute of
(N,N-Dimethylheptylamine N-oxide) were tested for protein              Technology, MC 210-41, 1200 E. California Blvd., Pasadena,
resistance and only heptylamine N-oxide exhibited resistance           CA, 91125
similar to TMAO. The principle of osmolyte exclusion from              We have been investigating the use of non-canonical
the protein backbone is responsible for the protein resistant          amino acids in antibody fragments in an effort to improve
property of the surface. We speculate that this novel process          the chemical and physical properties of these proteins. We
of surface modification may have wide application due to its           have incorporated aliphatic, azide, and alkyne analogs of
simplicity, low cost, regenerability and flexibility.


methionine (Met) into an anti-digoxin single chain variable             protein-folding motif, an α−helical coiled coil. The strategy
fragment (scFv) and have studied the properties of the                  relies on the use of cationic and anionic modules derived from
resulting proteins.                                                     the heptad repeats of polar (P) and hydrophobic (H) residues,
Our initial efforts have focused on studying the binding                PHPPHPP. Different fibrous morphologies ranging from
properties and affinity maturation of scFvs containing non-             individual fibers to porous matrices were shown to strongly
canonical amino acids. Using an E. coli cell surface display            correlate with the specific arrangements of the modules
system, we found that global replacement of Met with any of             indicating significant architectural flexibility of the design.[2]
the analogs of interest resulted in scFvs with lower display            Complementary to this is our recent design of self-assembling
levels and/or reduced binding affinity to a fluorescent digoxin         modules incorporating cell-adhesion motifs. The motifs are
probe. We therefore developed an error-prone PCR library                conformationally constrained mimetics of laminin adhesion
of antibody fragments and used flow cytometry to isolate                site YIGSR, the natural antagonist of the integrin-associated
functional, alkyne-containing scFvs. A frequently isolated              laminin receptor 1 (LAMR1). Observed differential
clone was tolerant of both Met and several non-canonical                responses during conformational screening of the mimetics
amino acids. Interestingly, the binding properties of this              using cell attachment assays revealed enhanced attachment
clone improved upon replacement of Met with an azide-                   activities for turn-like structures.[3] Combined the findings
containing amino acid. Screening of an additional library in            permit a functional design of self-assembling cell-responsive
several non-canonical amino acid contexts showed that that              matrices.
fragments could be affinity matured in each of these contexts.          1 Ulijn, R. V. and Smith, A. M.; Chem. Soc. Rev., 2008, 37,
Furthermore, the amino acid context appears to affect the                 664-675.
frequencies of particular mutations that occur in isolated
clones.                                                                 2 Ryadnov, M.G., Bella, A., Timson, S. and Woolfson, D.N.;
                                                                          J. Am. Chem. Soc., 2009, 131, 13240–13241.
These results indicate that scFvs containing non-canonical
amino acids are evolvable and can be affinity matured into              3 Bella, A., Lewis, H., Phu, J., Bottrill, A., Mistry, S., Pullar,
high affinity binders. Introducing the unique chemical                    C.E., and Ryadnov M.G.; J. Med Chem, 2009, 52, 7966–
functionalities present in non-canonical amino acids into                 7969.
binding proteins may provide distinct advantages in the
molecular recognition of foreign antigens. Furthermore,                 161
azide- and alkyne-containing scFvs may be highly effective              Flexible backbone design to optimize 2F5 mAb epitope
for performing chemical scFv modifications using “click                 transfer to heterologous proteins
chemistry.”                                                             Mihai Azoitei1, Jean-Philippe Julien2, Yih-En Ban1,
Acknowledgements: NIH, NDSEG (JAV), Beckman Institute,                  Alexandria Schroeter1, Oleksandr Kalyuzhniy1, Yumiko
Joseph J. Jacobs Institute for Molecular Engineering for                Adachi1, Steve Bryson2,4, David Baker1, Emil Pai3,4, William
Medicine                                                                Schief1
                                                                         Biochemistry, University of Washington, 1705 NE Pacific
160                                                                     St., HSB J-405, Seattle, WA, 98195, 2Biochemistry,
                                                                         Biochemistry, Medical Biophysics and Molecular Genetics,
Toward the Design of Self-Assembling Peptide Modules                    University of Toronto, 1 King’s College Circle, Toronto, M5S
Supporting Cell-Attachment and Growth                                   1A8, Canada, 4Division of Cancer Genomics and Proteomics,
Bella Angelo1, Christine Pullar2, Paul M. Cullis1, Maxim G.             Ontario Cancer Institute/Princess Margaret Hospital, 101
Ryadnov3                                                                College Street, MaRS/TMDT, Toronto, ON, M5G 1L7,
  Chemistry, University of Leicester, George Porter Building,           Canada
University Road, Leicester, LE1 7RH, United Kingdom,
  Cell Physiology and Pharmacology, University of Leicester,            Structure-based grafting of antibody epitopes onto scaffold
Henry Wellcome Building, University Road, Leicester, LE1                proteins is a novel platform for vaccine design and for
9HN, United Kingdom, 3Biotechnology and Bioengineering,                 testing the ability of computational design to manipulate
National Physical Laboratory, Hampton Road, Teddington,                 protein structure and protein-protein interactions. In this
London, TW11 0LW, United Kingdom                                        study we used the Multigraft protocol within the Rosetta
                                                                        protein modeling framework to transfer the linear epitope
Self-assembling peptide-based materials present perhaps                 of 2F5, a broadly neutralizing antibody against HIV-1,
the most straightforward route to biomimetics of the native             onto multiple different protein scaffolds. Fixed backbone
extracellular matrix (ECM) as cell-supporting scaffolds for             modeling, followed by flexible backbone optimization, was
regenerative medicine.[1]                                               employed to engineer new proteins, called epitope-scaffolds,
Recently, we have reported a modular approach to designing              that structurally stabilize the bound conformation of the 2F5
fibrous nano- to microscopic matrices based on a native                 epitope and could subsequently act as antigens.


In the first stage of computational procedure, the epitope             Carbohydrate microarray binding experiments confirmed
side-chains were transferred to non-HIV proteins that have             that the specificity profile of carbohydrate binding is nearly
stretches of exposed backbone similar to the backbone                  identical for all mutants. In particular, the mutants selectively
conformation of the 2F5-bound epitope (RMSD<1A).                       bound oligosaccharides containing the Manα(1→2)Man
Additional mutations were designed to eliminate unfavorable            linkage, which is the known minimal binding unit of CVN.
interactions. Over twenty epitope-scaffolds were identified,           We have also investigated: the effect of removing the
designed and experimentally characterized. The resulting               native disulfide bridges of CVN on protein stability; the
epitope-scaffolds differ from the parent proteins by 10                consequences of engineering new disulfide bonds into the
mutations on average and most of them bound 2F5 with                   CVN fold; and the effects of key binding site mutants on both
affinities in the same range or better than the corresponding          stability and carbohydrate recognition.
epitope peptides.
However, some epitope-scaffolds had lower affinities for 2F5           163
than predicted and we hypothesized that in these cases the
epitope was stabilized in a non-optimal conformation. We               Towards a molecular understanding of protein solubility
employed flexible backbone modeling to optimize structural             Ryan Kramer1, Saul Trevino2, C. Nick Pace1,2, J. Martin
mimicry on four such epitope-scaffolds. In this procedure,             Scholtz1,2
the epitope backbone replaced the native scaffold backbone
                                                                        Biochemistry & Biophysics, Texas A&M University, 440
at the exact positions where only the epitope side chains were         Reynolds Medical Building, College Station, TX, 77843-
previously transferred. Loop modeling and design was used              1114, 2Molecular and Cellular Medicine, Texas A&M Health
to connect the epitope backbone to the parent scaffold. It             Science Center, 440 Reynolds Medical Building, College
was hoped that this procedure would ensure that the bound              Station, TX, 77843-1114
conformation of the epitope could at least be induced upon             Understanding how to enhance protein solubility is of great
2F5 binding. For two of the redesigned epitope-scaffolds, this         interest to crystallographers, the pharmaceutical industry,
procedure identified sequence changes that led to considerable         and other protein chemists. However, methods to directly
improvement in 2F5 binding, from a KD of 633nM to 9nM                  measure protein solubility often lead to the formation of
and 41nM to 10nM respectively. Ongoing crystallographic                super-saturated solutions, gels, or protein crystallization.
studies will test the accuracy of the computational modeling.          One method to get around this is to lower the solubility of
The results demonstrate that flexible backbone methods for             a protein using an extraneous agent such as polyethylene
epitope grafting can in some cases dramatically improve                glycol, salt, or an alcohol. When introducing an extraneous
binding affinities over those achieved by fixed backbone               agent to precipitate protein solutions, it is necessary to be sure
methods alone.                                                         that the protein is folded under experimental conditions and
                                                                       that the measured solubility value are not initial concentration
162                                                                    dependent. We have a model system (Ribonuclease Sa) and
                                                                       have put all 20 amino acids at a completely solvent exposed
Stabilization of Cyanovirin-N, a Potent HIV-inhibiting                 position to create an amino acid solubility scale in the
Lectin: Computer-aided Design and Experimental                         context of a protein. We have measured the relative solubility
Characterization                                                       of these mutants using ammonium sulfate, polyethylene
Vadim Patsalo1, Daniel P. Raleigh2,3, David F. Green1,3                glycol, and isopropanol as precipitants. We find that charge
  Applied Math and Statistics, Stony Brook University, Math            plays a significant role in modulating protein solubility, and
Tower, Stony Brook, NY, 11794-3600, 2Chemistry, Stony                  we are further investigating this by constructing mutants
Brook University, Stony Brook University, Stony Brook,                 of Ribonuclease Sa with increased surface charge and by
NY, 11794-3400, 3Graduate Program in Biochemistry and                  measuring protein solubility as a function of pH
Structural Biology, Stony Brook University, Stony Brook
University, Stony Brook, NY, 11794-3600
Cyanovirin-N (CVN) is a 12-kDa pseudo-symmetric
cyanobacterial lectin that has been shown to inhibit infection
by the Human Immunodeficiency Virus (HIV) by binding to
high-mannose oligosaccharides on the surface of the viral
glycoprotein gp120. Through rational and computational
approaches, we have been able to identify CVN mutants
that stabilize the protein fold. By mutating the buried polar
sidechains of Ser11, Ser20, and Thr61 to aliphatic groups, we
stabilized CVN by nearly 12°C against thermal denaturation,
and by 1M of GuaHCl against chemical denaturation.


164                                                                        The transplantation of a complex epitope or binding site from
                                                                           one protein of known structure to other scaffold proteins
Oligonucleotide Recombination for Combinatorial                            serves as a challenging test for flexible backbone protein
Optimization of Yeast Metabolic Flux for Terpenoid                         design and could be employed to devise novel vaccines or
Production                                                                 binding reagents. We sought to graft a complex antibody
Nili Ostrov1, Parayil Kumaran Ajikumar2, Zachary Fowler3,                  epitope involving two non-contiguous segments from the HIV
Mattheos Koffas3, Gregory Stephanopoulos2, Virginia                        gp120 envelope protein onto a heterologous scaffold protein.
Cornish1                                                                   Within Rosetta, we developed a general computational
  Department of Chemistry, Columbia University, 3000                       method called Multigraft for transplantation of complex
Broadway, New York, NY, 10027, 2Department of Chemical                     epitopes. Given an input structure of an antibody-epitope
Engineering, Massachusetts Institute of Technology, 77                     complex, Multigraft searches the PDB for suitable acceptor
Massachusetts Avenue, Cambridge, MA, 02139, 3Department                    scaffolds using geometrical criteria, replaces the backbone
of Chemical and Biological Engineering, University at                      of the candidate scaffolds with the epitope backbone, and
Buffalo, The State University of New York, 904 Furnas Hall,                employs iterative loop modeling and design to build a
Buffalo, NY, 14260                                                         continuous backbone. Starting from a crystal structure of the
The plasticity of the metabolic network makes microorganism-               b12 antibody in complex with gp120, a number of scaffolds
based biosynthesis of natural products a promising platform                were designed, produced and their affinity for b12 assayed
for rapid, efficient and highly versatile drug and commodity               by SPR. In that first generation, only one scaffold had a
production. A major bottleneck to metabolic engineering,                   detectable and specific affinity for b12, but the KD (≈300uM)
however, remains the inability to extensively modify metabolic             was significantly higher than the KD for gp120 (20nM). To
flux in the host strain to achieve high yields of the natural              improve b12 binding, computation-guided evolution was
product. Because numerous enzymes must be targeted, their                  undertaken. Cycles of conformational sampling and design
expression levels attenuated to different degrees, and changes             were performed to diversify the sequence of the connecting
are often synergistic and have effects that are difficult to               regions between the epitope segments and the acceptor
predict precisely, this optimization process is iterative, labor-          protein. Directed mutagenesis libraries were derived from
intensive and costly. Thus, building on the observation that               the resulting sequences and were screened for b12 binding
homologous recombination in yeast is highly efficient, here                using yeast surface display. The best clone bound b12 with
we exploit oligonucleotide mutagenesis as a straightforward                a KD of 35nM, comparable to the KD for the b12-gp120
and general technology for combinatorial mutagenesis of                    interaction. To our knowledge, this is the first successful
host metabolic flux. We present model studies optimizing                   backbone transplantation of a discontinuous binding site to
oligonucleotide recombination for library mutagenesis of                   a heterologous protein. Further, the approach shows how to
chromosomal genes based on repair of nonsense codons                       leverage aggressive computational design with an efficient in
in auxotrophic markers. We present progress towards the                    vitro evolution strategy. Lastly, the optimized scaffold could
application of these tools to optimize metabolic flux in S.                be useful for HIV vaccine research to detect or elicit b12-like
cerevisiae for terpenoid production. Together these results                antibodies.
establish oligonucleotide recombination as a simple and                    B. C. and M. A. contributed equally to this work.
powerful library mutagenesis technique and advance our
efforts to engineer the cell for fully in vivo directed evolution.         166
More broadly, our network-oriented experimental approach
expands the toolkit available for engineering living cells                 Structural Analysis of Computationally Designed Kemp
toward the routine production of valuable natural products                 Elimination Catalysts
in yeast.                                                                  Toni M. Lee1, Heidi K. Privett2, Stephen L. Mayo1
                                                                            Biochemistry and Molecular Biophysics Option, California
165                                                                        Insititute of Technology, 1200 E. California Blvd. MC 114-
                                                                           96, Pasadena, CA, 91125, 2Protabit LLC, Protabit LLC, 201
Transplantation of a complex binding site using                            S. Lake Ave, Suite 507, Pasadena, CA, 91101
computational design and in vitro evolution
Bruno Correia1,2, Mihai Azoitei1, Yih-En Ban1, Oleksandr                   The de novo creation of novel enzymes constitutes
Kalyuzhniy1, Alexandria Schroeter1, David Baker1, William                  an established goal of the biochemical and industrial
Schief1                                                                    communities. Traditional methods of enzyme engineering,
  Biochemistry, University of Washington, 1705 NE Pacific                  however, require the pre-existence of a natural catalyst for
St.1, HSB J-513, Box 357350, Seattle, WA, 98195, 2Instituto                the reaction of interest. We have employed a computational-
de Tecnologia Quimica e Biologica, Universidade Nova de                    based methodology to generate enzymes that catalyze the
Lisboa, Av. da Republica, Estacao Agronomica Nacional,                     Kemp elimination, a reaction for which no natural catalyst
Oeiras, 2780-157, Portugal                                                 exists. The Kemp elimination of 5-nitro benzisoxazole is a


heavily studied proton transfer reaction. Crystal structures of         stabilize the interaction of the two mutated partner proteins.
the Kemp elimination catalytic antibody 34E4 demonstrate                Experimentally, we used an array of different assays to verify
catalysis occurs through three features: (1) a general base             the interaction of the designed proteins, including a high-
to remove the proton from the substrate, (2) an aromatic                throughput mant-GDP [2’-/3’- O- (N’- Methylanthraniloyl)
residue to hydrophobically stack with the substrate, and (3)            guanosine- 5’- O- diphosphate] binding assay, a GDP/mant-
a hydrogen bond donor to stabilize the developing phenolic              GDP exchange assay, an in vitro effector recruitment assay
charge during ring opening. We have obtained three structures           and cell-based polarization assays.
using X-ray crystallographic methods for two active designs             Results and Conclusions: Directed by computational design
generated using computational methods, HG-2 and 1A53-2.                 applying ROSETTA, a Cdc42-ITSN mutant pair was
This structural evidence validates that each design consists of         designed and experimentally validated. The details of the
the aforementioned catalytic residues grafted onto a general            designed interaction were characterized by a crystal structure
TIM barrel scaffold. HG-2 and 1A53-2 were co-crystallized               of the designed pair of Cdc42-ITSN mutants. Future work
with the substrate analog 5-nitro benzotriazole (NBT). In               involves generalizing the design protocols and applying
each case, the substrate clearly maintains the main contact             them to other GTPase interactions. Designing orthogonally
to the catalytic aspartate base. A third structure of 1A53-2            interacting protein pair may be an effective way to modulate
in apo form suggests active site reorganization must occur              and control cellular processes, which could be generalized in
before ligand binding. These results provide structural                 different systems and achieved quickly with the benefits of
evidence of catalytically relevant enzyme-substrate binding             computational design protocols as well as high-throughput
in a computationally designed protein catalyst.                         experimental assays.
This work was made possible through funding from the
National Science Foundation.                                            168

167                                                                     Towards design of species-specific snake toxins
                                                                        Oz Sharabi, George Dakwar, Yonatan Aizner, Julia Shifman
Reengineering Small GTPase Interactions Using                           The Hebrew University, Givat Ram, Jerusalem, Israel
Computational Design to Rewire Protein Signaling                        Predicting mutations that enhance protein–protein affinity
Pathways                                                                remains a challenging task, especially for high-affinity
Sen Liu1, Gregory Kappa2, Noah Ollikainen2, Tanja                       complexes. We developed improved computational methods
Kortemme3                                                               that can more accurately model important intermolecular
  UCSF/UCB Center for Engineering Cellular Control                      interactions observed in high-affinity protein-protein
Systems, University of California, San Francisco, 1700 4th              complexes. Performance of the improved methods was tested
Street, Byers Hall 309A, San Francisco, CA, 94158, 2UCSF/               on a complex between a synaptic enzyme acetylcholinesterase
UCB Center for Engineering Cellular Control Systems,                    (AChE) and its inhibitor snake toxin fasciculin (Fas). Fas
University of California, San Francisco, 1700 4th Street,               binds with pM affinity to AChE from Humans (hAChE),
Byers Hall 309A, San Francisco, CA, 94158, 3UCSF/UCB                    Torpedo Californica (tAChE), and similar species. We
Center for Engineering Cellular Control Systems, University             redesigned the Fas binding interface to enhance its affinity
of California, San Francisco, 1700 4th Street, Byers Hall               for tAChE. A number of computationally designed Fas
308E, San Francisco, CA, 94158                                          mutants were experimentally constructed and tested for
Study objective: To design orthogonal interacting protein               their ability to bind to and inhibit the enzyme. Our best Fas
pairs of small GTPases that could be used to control cell               mutant exhibited a 1.2 kcal/mol improvement in the binding
movement.                                                               free energy, proving that enhancing affinity of evolutionary
Background and Methods: Cdc42-ITSN (Cell division                       optimized complexes is still possible. Interestingly, several
control protein 42 homolog and Intersectin) and Rac1-Tiam1              Fas mutants optimized for binding to tAChE showed
(Ras-related C3 botulinum toxin substrate 1 and T-lymphoma              considerably decreased affinity to hAChE, demonstrating the
invasion and metastasis 1) are important interacting protein            possibility of designing a species-specific snake toxin. This
pairs involved in cell polarization, chemotaxis and movement.           leads us to the next challenge, design of Fas that can bind to
We aimed to design and validate mutant Cdc42-ITSN and                   AChE from Drosophila (dAChE) but not to hAChE. While
Rac1-Tiam1 orthogonal pairs through a combination of                    dAChE is very similar to hAChE both at the sequence and the
computational and experimental methods. Computationally,                structure level, the Fas binding site on dAChE has evolved to
the ROSETTA program (http://www.rosettacommons.                         loose its geometric complementarity to the toxin and hence
org) was used to predict and redesign key interactions                  no binding is observed in nature. To restore this geometric
in the interface. To engineer orthogonality, we designed                complementarity between the two proteins, we first created
mutations to destabilize the interaction between a wild-type            a Fas chimera, by substituting one loop on Fas with a loop
protein and a mutant protein of its wild-type partner, but to           from a homologous toxin. We then docked the chimera Fas


onto dAChE using the recently developed Dock-for-Design                 170
procedure and finally redesigned Fas for best interactions
with dAChE. Our computational results are currently being               Oxidative Assembly of the Octadecameric Complex of an
tested in the laboratory by expressing the Fas mutants and              Adipocyte Hormone Requires Oxygen and Stabilization
assaying them for binding to dAChE and hAChE.                           of Assembly Intermediates by Disulfide Bonds – A Model
                                                                        for Assembly of Protein Complexes with Short Collagen
Protein Folding: General Aspects                                        Tsu-Shuen Tsao1, David Briggs1, Martha Nunez1, Andrew
(169 – 182)                                                             1
                                                                         Chemistry and Biochemistry, University of Arizona,
                                                                        Medical Research Building, 1656 E Mabel St., Tucson, AZ,
169                                                                     85724, 2Chemistry and Biochemistry, University of Arizona,
FoldEco: simulating protein folding in E. coli                          Biological Sciences West, 1041 E Lowell St., Tucson, AZ,
Kristine Pobre1, Evan Powers2, Lila Gierasch1                           85721
  University of Massachusetts, 710 North Pleasant St.,                  Adiponectin is an adipocyte hormone that primarily homo-
Amherst, MA, 01003, 2The Scripps Research Institute, 10550              oligomerizes into trimeric, hexameric and octadecameric
North Torrey Pines Road, La Jolla, CA, 92037                            species. Small amounts of nonameric and dodecameric
The interplay of the various networks of reactions that affect          forms could also exist, suggesting a stepwise assembly
protein folding, misfolding, aggregation, and degradation in            mechanism. The basic trimer consists of a globular head
the Escherichia coli (E. coli) cell is complex--challenging             and a short triple-helical collagen tail with only 22 repeats.
experimental strategies are used to interrogate it. Many                Electron micrographs of adiponectin octadecamers revealed
players including chaperones, disaggregases, and degradatory            a bouquet-like structure composed of 6 trimers with extensive
enzymes are involved, and together they perform crucial                 interactions only in the N-terminal half of the collagen
roles in maintaining the cell’s homeostasis. We are utilizing           tails. We previously showed that assembly of octadecamers
an integrated computational and experimental approach                   requires the pairing of conserved cysteine residues on adjacent
to study the effects of the various cellular networks on                monomers near the N-terminus into disulfide bonds, but that
folding and aggregation. To this end, we have developed                 the mature complex itself is stable in the absence of any
FoldEco, which simulates processes affecting the balance of             covalent interactions. We hypothesized that the role of the
folding and aggregation in E. coli, and we are testing this             disulfide bonds is to stabilize assembly intermediates lacking
model experimentally. We have explored many conditions                  a critical threshold of collagen-mediated interactions. Here
that simulate those utilized in the laboratory, and used the            we present additional evidence to support that hypothesis.
simulations to guide further conditions to test experimentally.         Isolated hexamers which are fully oxidized failed to
We are utilizing cellular retinoic acid binding protein (CRABP)         reassemble, but reduction of a small percentage of disulfides
and aggregation-prone variants as a system to monitor the               within hexamers permitted reassembly into higher oligomers,
balance of folding and aggregation. For example, FoldEco                indicating that free protein thiols can catalyze transthiolations
predicts that an increase in the degradation rate results in a          which result in higher assembly states. Furthermore, kinetic
decrease in the aggregation propensity. We are comparing                analysis showed that the rate-limiting step in reassembly of
the yield of folded native protein versus aggregate in the              octadecamers is the availability of free protein thiols rather
presence and absence of lon protease in E. coli. Similarly,             than interaction between collagen domains. We also showed
we are exploring the impact of GroEL/ES and DnaK cellular               that oxidative assembly of adiponectin can utilize oxygen or
concentrations on the balance of folded and aggregated                  hydrogen peroxide as the electron acceptor. While hydrogen
CRABP. To extend these findings, we are exploring how                   peroxide accelerated reassembly, chelation of ions that could
FoldEco predicts chaperone dependence of a set of model                 generate radicals only partially inhibited reassembly. In
proteins with different stabilities, chaperone affinities, and          contrast to oxygen or hydrogen peroxide, oxidized DTT could
misfolding propensities. Preliminary results suggest that               not serve as an electron acceptor in reassembly reactions.
GroEL can rescue slow folders well, but DnaK is beneficial              Taken together, our results suggest a “cystine ratchet” model
to misfolders.                                                          for assembly of large protein complexes with short collagen
                                                                        domains, in which assembly intermediates are stabilized by
                                                                        disulfide bonds until a critical mass of collagen domains have
                                                                        assembled to a level that can sustain itself.


171                                                                    outer surface of the barrel (Fig. 1) [1]. Moreover, we reported
                                                                       that under an acidic condition (pH 4.0), the thermal unfolding
Folding the circular permutants of IL-1β: A balancing                  of mouse L-PGDS is a completely reversible and follows a
act between topological symmetry and functional                        three-state unfolding process, including an intermediate state
heterogeneity                                                          (I) between the native state (N) and the unfolded state (U)
Dominique Capraro1, Shachi Gosavi2, Melinda Roy1, Jose                 [2]. The transition temperatures (Tm) of N ↔ I and of I ↔ U
Onuchic3, Patricia Jennings1                                           were 46.9 °C and 60.8 °C, respectively [2]. In this study, we
  Chemistry and Biochemistry, UCSD, 9500 Gilman Drive,                 investigated the thermal unfolding process by NMR and CD
NSB 3110, Mail Code 375, La Jolla, CA, 92093, 2National                spectroscopy in order to characterize the intermediate state of
Centre for Biological Sciences, TIFR, GKVK, Bellary Road,              mouse L-PGDS.
Bangalore, 560065, India, 3Center for Theoretical Biological
Physics, UCSD, 9500 Gilman Drive, Urey Hall 710, Mail                  [Methods] Mouse L-PGDS contains two Trp residues at
Code 374, La Jolla, CA, 92093                                          positions 43 and 54. The Trp43 residue is located at the
                                                                       bottom region of the β-barrel, whereas the Trp54 residue
The three-fold pseudo-symmetric cytokine, IL-1β, has three             locates at the upper region of the β-barrel and faces the
possible trefoil folding nuclei that generate three symmetry-          outside of the β-barrel (Fig. 1). We purified two kinds of
related routes whose contribution to overall folding can               proteins, L-PGDS and W54F-L-PGDS which contains only
be modulated by sequence variations in the individual                  a single Trp residue (Trp43). For NMR and CD experiments,
trefoils. Only two of these nuclei and their routes have been          the protein concentrations were adjusted to 0.3 mM and 0.05
observed. Here, we explore the effect of the energetic and             mM, respectively. All experiments were performed in 20 mM
topological heterogeneity of the trefoils on the likelihood            sodium acetate buffer (pH 4.0).
of their routes using carefully designed circular permutants
and a combination of kinetic experiments and simulations.              [Results] Thermal unfolding of L-PGDS was monitored by
Our experiments show that the rate and region of initial
                                                                        H-15N HSQC spectra. With increasing temperature from 25
nucleation of all permutants is similar to WT but further              °C to 45 °C, some cross-peaks shifted and intensity of some
folding of the partially-structured ensemble is always slower.         other peaks decreased with signal broadening. The residues
The WT energetic distribution appears to be sufficient to              displaying large chemical shift change on the 1H-15N HSQC
let the WT-route dominate. Folding simulations supports                spectrum were located near aromatic side-chains, suggesting
these experimental conclusions; wherein permutants cut in              changes in the side-chain interactions of L-PGDS. Further,
the second-trefoil can switch their route to the entropically-         some signals disappeared on the 1H-15N HSQC spectrum,
unfavorable WT-route when aided by a slight energetic bias             and these missing residues were located in the upper region
towards the WT-nucleus. More generally, circular permutation           of the β-barrel, indicating conformational changes in the
can create a mismatch between energetics and topology in               region. At 54 °C, at which the intermediate state is maximally
proteins where functional topological complexity has been              accumulated [2], the 1H-15N HSQC spectrum of L-PGDS
compensated by evolving local energetic stabilization.                 showed limited chemical shift dispersion and severe signal
                                                                       broadening except for the signals of the residues located at
                                                                       the bottom region of the β-barrel. These results indicated
                                                                       that the bottom region of β-barrel is still maintained in the
NMR and CD analysis of an intermediate state in the                    intermediate state.
thermal unfolding process of mouse lipocalin-type
prostaglandin D synthase
Yuya Miyamoto1,2, Yasuo Noda3, Shigenori Nishimura1,
Shin-ichi Segawa3, Takashi Inui1
  Graduate school of Life and Environmental Sciences,
Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku,
Sakai-shi, Osaka, 599-8531, Japan, 2Research Fellow of
the Japan Society for the Promotion of Science, 8, Ichiban-
cho, Tiyoda-ku, Tokyo, 102-8472, Japan, 3School of Science
and Technology, Kwansei Gakuin University, 2-1, Gakuen,
Sanda, Hyogo, 669-1337, Japan                                          In order to probe the local conformational changes of
[Introduction] Lipocalin-type prostaglandin D synthase                 L-PGDS during N ↔ I transition, we monitored the thermal
(L-PGDS) is a multi-functioning protein in the lipocalin               transitions of Trp54 (upper region) and Trp43 (bottom
superfamily, acting as a PGD2-synthesizing enzyme and as               region) by 1D NMR and CD spectroscopy, respectively. By
an extracellular transporter for small hydrophobic molecules.          1D NMR measurements, the Tm for the thermal transition of
L-PGDS has a typical lipocalin fold with an eight-stranded             Trp54 of L-PGDS was calculated to be 41.4 °C using a two-
antiparallel β-barrel and a long α-helix associated with the           state equilibrium unfolding model. By CD measurement at


290 nm, the Tm for the thermal transition of Trp43 of W54F-             174
L-PGDS calculated to be 47.5 °C and was in good agreement
with that for N ↔ I transition of L-PGDS (46.9 °C) [2].                 Investigation of the Thermal Stability of a Y-Family DNA
[Conclusions] The conformation of the upper region of the               Shanen Sherrer1, Lindsey Pack1, Kevin Fiala1, Jason
β-barrel, where Trp54 is located, changes at the temperature            Fowler1, Zucai Suo2
lower than that of the bottom region of the β-barrel, where             1
                                                                         Biochemistry, The Ohio State University, Room 822
Trp43 is located. In the intermediate state in the thermal              Biological Science Building, 484 W. 12th Ave, Columbus,
unfolding of L-PGDS, the bottom region of L-PGDS is still               OH, 43210, 2Biochemistry, The Ohio State University,
maintained.                                                             Room 880 Biological Science Building, 484 W. 12th Ave,
1 Miyamoto, Y. et al. (2010) J. Struct. Biol. 169, 209-218.             Columbus, OH, 43210
2 Iida, T. et al. (2008) FEBS J. 275, 233-241.                          Sulfolobus solfataricus DNA Polymerase IV (Dpo4) is the
Fig. 1. Solution structure of mouse L-PGDS (PDB code                    most studied member of the Y-family DNA polymerases.
2RQ0).                                                                  The physiological temperature of Sulfolobus solfataricus is
                                                                        80°C. However, Dpo4 is commonly used in kinetic studies
173                                                                     conducted at 37°C and crystallization studies performed
                                                                        under ambient conditions due to experimental limitations
Physical-chemical determinants of coil conformations in
                                                                        and relevance to similar enzymes in humans. With such a
globular proteins
                                                                        discrepancy in temperature, the in vivo relevance of these
Lauren Perskie1, George Rose2
                                                                        kinetic and structural studies on Dpo4 has been a persistent
  Biophysics, Johns Hopkins University, 3400 N. Charles
                                                                        question. In this study, we have employed a circular
St., Baltimore, MD, 21218, 2Biophysics, Johns Hopkins
                                                                        dichroism spectroscopic investigation on the secondary
University, 3400 N. Charles St, Baltimore, MD, 21218
                                                                        structural changes of Dpo4 over a temperature range from
We present a method with the potential to generate a library            26°C to 119°C and when Dpo4 forms a binary complex with
of coil segments from first principles. Proteins are built              DNA. We determined that both Dpo4 and DNA underwent
from α-helices and/or β-strands interconnected by these                 significant structural changes as Dpo4 bound to DNA at 26°C.
coil segments. Here, we investigate the conformational                  We also discovered that Dpo4 had a three-state cooperative
determinants of short coil segments, with particular                    unfolding trend with an extremely high melting temperature
emphasis on chain turns. Toward this goal, we extracted a               characteristic of hyperthermophiles. In addition, we
comprehensive set of 2-, 3- and 4-residue turns from X-ray              demonstrated through a series of thermal denaturation studies
elucidated proteins and classified them by conformation. A              that the linker region between the little finger and the thumb
remarkably small number of unique conformers account for                domains of Dpo4 was the source of structural instability and
most of this experimentally-determined set, while remaining             that Dpo4’s individual domains were thermostable. Most
members span a large number of rare conformers, many                    importantly, the evidence suggested that the Dpo4 secondary
occurring only once in the entire protein database. Factors             structure at 37°C was similar to the Dpo4 secondary structure
determining conformation were identified via Metropolis                 at 80°C. Thus, it is structurally relevant to conduct kinetic
Monte Carlo simulations devised to test the effectiveness of            studies at 37°C when using Dpo4 as the model Y-family
various energy terms. Simulated structures were validated               DNA polymerase.
by comparison to experimental counterparts. After filtering
rare conformers, we found that 98% of the remaining                     175
experimentally-determined turn population could be
reproduced by applying a hydrogen-bond energy term to an                Structural Characterization of the Sub-millisecond
exhaustively generated ensemble of clash-free conformers                Refolding Intermediate of CheY
in which no backbone polar group lacks a hydrogen-bond                  Sagar Kathuria, Osman Bilsel, Paul Nobrega, C Robert
partner. Further, at least 90% of longer coil segments, ranging         Matthews
from 5- to 20-residues, were found to be structural composites          Department of Biochemistry and Molecular Pharmacology,
of these shorter primitives. These results are pertinent to             University of Massachusetts Medical School, 364 Plantation
protein structure prediction, where approaches can be divided           St, Worcester, MA, 01605
into either empirical or ab initio methods. Empirical methods           CheY is a small, single-domain protein, comprising five
use database-derived information; ab initio methods rely                repeating βα elements that are organized in a α/β/α sandwich
on physical-chemical principles exclusively. Replacing the              topology.     Its equilibrium denaturation properties are
database-derived coil library with one generated from first             consistent with a two-state folding mechanism; however,
principles would transform any empirically-based method                 its kinetic folding properties are more complex. A sub-
into its corresponding ab initio homologue.                             millisecond burst-phase intermediate is observed during
Funding source: The Mathers Foundation.                                 refolding, followed by the slow trans-cis isomerization of a


conserved cis prolyl bond. Unfolding reveals a partitioning               177
between isomerization and unfolding that varies with the final
urea concentration. The early refolding intermediate displays             Probing the structure of the H2A-H2B dimeric kinetic
considerable native-like secondary structure and stability, and           folding intermediate by site-directed mutagenesis
this species has been shown by both coarse-grained simulations            Paul Guyett, Lisa Gloss
and experiments to be an obligate kinetically-trapped species.            School of Molecular Biology, Washington State University,
Current thinking posits that the rapid formation of a small but           Biotechnology/Life Sciences Room 140A, 1715 NE South
locally-connected cluster of branched aliphatic side chains on            Fairway Rd., Pullman, WA, 99164-7520
one side of the β-sheet results in a kinetic trap that precludes          We are using the eukaryotic histone heterodimer, H2A-H2B,
access to the transition state. The kinetic coupling of the               to study the formation of both intra- and inter-molecular
hydrophobic collapse with the development of secondary /                  contacts during oligomeric protein folding. Each monomer
tertiary structure are being probed with a micro-fluidic mixer            contains a histone fold, which consists of a long central
interfaced to a host of spectroscopic techniques including                α-helix flanked by two shorter α-helices connected by
time resolved fluorescence, circular dichroism, small angle               short loops; the majority of the dimer interface is along the
X-ray scattering and Förster resonance energy transfer.                   central α-helix. The kinetic folding mechanism of H2A-H2B
Such kinetically-trapped intermediates with native-like                   involves the rapid folding and association of the unfolded
secondary structure have been observed in other βα-repeat                 monomers to a partially folded dimeric intermediate, which
proteins, including TIM barrels and several other members                 further folds to the native dimer in the rate-determining step,
of the CheY family. A detailed analysis of the structure of               i.e. 2U → I2 → N2. To determine what residues are important
these intermediates will help in understanding the role of                to the structure and stability of the dimeric intermediate,
sequence in dictating off-pathway folding mechanisms in                   large, hydrophobic, buried residues along the dimer interface
βαrepeat proteins. This work was supported by NSF grants                  as well as residues that make predominantly intra-molecular
MCB0327504 and MCB0721312 awarded to CRM.                                 contacts were mutated to alanine. Most mutations reduced
                                                                          the dimer stability from 15.2 kcal mol-1 to ~11.5 kcal mol-1,
176                                                                       without significantly altering the m-value of 5.0 kcal mol-1M-1.
                                                                          Kinetic studies are in progress to determine if these residues
Slipknotting upon Native-like Loop Formation in a Trefoil                 are structured in the I2 ensemble. Analysis of these mutations
Knotted Protein                                                           will help elucidate how H2A-H2B forms concerted intra- and
Jeffrey Noel, Joanna Sulkowska, Jose’ Onuchic                             inter-molecular interactions along the folding pathway.
UC San Diego, MC 0374, 9500 Gilman Drive, La Jolla, CA,
92092                                                                     178
We map the free energy surface of the smallest known
knotted protein with an all-atom structure-based protein                  Dissecting the CL domain of streptococcal collagen-like
model. Experimental data suggests that knotted proteins are               protein 2
able to fold without any additional help, however current                 Zhuoxin Yu1,2, Barbara Brodsky1, Masayori Inouye1
experimental resolution is unable to monitor knot formation
                                                                           UMDNJ-Robert Wood Johnson Medical School, Department
during folding. Energy landscape theory can be employed to                of Biochemistry, Piscataway, NJ, 08854, 2UMDNJ-GSBS,
probe the microscopic dynamics of folding knots. Our results              675 Hoes Lane, Piscataway, NJ, 08854
show that, due to the topological constraint, the protein folds           Streptococcal collagen-like protein 2 (Scl2) forms a stable
through a three state mechanism that contains (i) the precise             triple-helix despite the absence of hydroxyproline (Hyp), the
nucleation site which pre-forms a correctly twisted native loop           low content of Pro, and the abundance of charged residues.
(first barrier) and (ii) a rate-limiting free energy barrier that         Here, the high yield cold-shock expression system was used to
is traversed by two parallel knot forming routes. The main                obtain recombinant Scl2 (termed V-CL) from Streptococcus
route corresponds to a slipknot conformation and the minor                pyogenes which contains an N-terminal variable domain V
pathway to an entropically limited plug motion. The knot                  followed by the collagen-like domain CL. It was found by
formation is a late transition state process and results show             circular dichroism (CD) that proper refolding of V-CL in
that non-specific shallow knots are a very rare and unstable              vitro requires the trimerization domain V. However, de novo
set of configurations both at and below folding temperature.              folding of the CL domain alone in E.coli does not require the
To connect with previous coarse-grained studies, we                       trimerization domain at either end, indicating the presence of
compared the results to those of a C_alpha-bead model. The                a nucleation region for proper folding within the CL domain.
comparison shows how specific side-chain packing smoothes                 In order to identify the nucleation domain, the CL domain
the complex topological landscape of a knotted protein.                   was dissected into three equal-size subdomains, A, B and
                                                                          C, and each of them was characterized. Biochemical and
                                                                          biophysical properties of A, B and C are quite different. The
                                                                          very N-terminal subdomain A is not resistant to trypsin while


both B and C are resistant. Tm of B is 4 oC higher than A and             structures within the E.coli ribosomal folding zone. Moreover,
C, and B is the only subdomain which can refold by itself.                the influence of prokaryotic ribosome on the poly-alanine
Subdomain C but not A or B aggregates heavily. Next we                    compaction was considered to be different from the one in
further compared these subdomains and examined whether                    eukaryotic system.
thermal stability is correlated to the length by constructing             [Supported by National Science Council: 97-2113-M-001-
tandem repeats of A, B and C. We also studied the roles of                003-MY2 and Academia Sinica]
Pro residues in subdomain C, which contains only seven Pro
all of which are at Y position. The replacement of these Pro              180
with Hyp or other residues substantially reduced the thermal
stability. These results suggest that the domain structure of             Beyond the test tube: Monitoring protein folding in vivo
the collagen from S. pyogenes has been optimized for folding              Linda Foit1, Antje Mueller1, Tsinatkeab Tadesse1, Gareth
and stability during evolution. This information may also                 Morgan2, Maximilian Kern1, Annekathrin von Hacht1, Lenz
help us design recombinant bacterial collagens with different             Steimer1, James Titchmarsh2, Stuart Warriner2, Sheena
properties to be utilized in biomaterial application.                     Radford2, James Bardwell1,3
                                                                           MCDB, University of Michigan, 830 N. University, Ann
179                                                                       Arbor, MI, 48109, 2Astbury Centre for Structural and
                                                                          Molecular Biology, University of Leeds, Garstang Building,
The possible folding zone within the E.coli ribosomal exit                Leeds, LS2 9JT, United Kingdom, 3Howard Hughes Medical
tunnel                                                                    Institute, 4000 Jones Bridge Road, Chevy Chase, MD, 20815
Ku-Feng Lin1, Chia-Sui Sun2, Yi-Chen Huang1, Jen-Tse
Huang1                                                                    Despite the increasing knowledge about factors affecting
  Institute of Chemistry, 2Institute of Biomedical Science,               protein stability, the question of how protein stability is
Academia Sinica, 128 Academia Road, Section 2, Nankang,                   determined in vivo still remains a fundamental question.
Taipei, Taiwan                                                            Previously, we have developed a powerful genetic system
                                                                          that directly links the stability of a protein in vivo to antibiotic
Previous studies have indicated that ribosomal exit tunnel                resistance (Foit et al. (2009), Mol Cell, 35, 861-871). In
is more than a passive environment. It can also promote                   this, the stability of a protein inserted into β-lactamase
the folding, regulation of protein synthesis and trafficking.             directly influences the resulting level of antibiotic resistance
From the chemical-pegylation experiments, the tunnel of                   conferred by the fusion. Our method provides a quantitative
eukaryotic ribosome has been shown to be highly refined and               output to monitor the stability of proteins in vivo. By
may exhibit different folding impacts on nascent peptides                 selecting for increased antibiotic resistance in vivo, we
within its different regions. In spite of the general comparison          used the system to isolate variants of a model protein that
between eukaryotic and prokaryotic ribosome have suggested                are stabilized thermodynamically and kinetically in vitro.
the structures are quite similar, the composition as well as              The ability to evolve protein stability in the absence of
functional assays has shown the tunnel may varies among                   functional requirements allows us to dissect the influences of
species and thus resulted in various kinds of ribosome-                   the selection pressures function and stability during protein
mediated interaction, e.g. SRP recruitment.                               evolution.
Here, we have tried to explore the influence of E.coli                    Recently, we utilized the system to identify chemical
ribosomal exit tunnel to the conformation of nascent peptide.             compounds that act as chemical chaperones in vivo. Here, a
Three green fluorescent protein (GFP) mutants harboring a                 poorly folding protein or peptide is inserted into β-lactamase
poly-alanine sequence were designed as sensors to detect                  and expressed in cells that are cultivated in media
the helix compaction preference in the tunnel. Site-specific              supplemented with chemical compounds. The addition of
fluorescent labeling was conducted by incorporation of                    substances known to stabilize proteins in vitro significantly
fluorophore-labeled aminoacylated tRNA into in vitro                      increases the expression level of the tripartite fusion in vivo
transcription/translation system. By applying the truncated               and the β-lactamase activity in cell extracts. The system can
mRNA, ribosome-nascent peptide complexe (RNC) with                        be used to screen for potential drugs preventing aggregation/
desired length was generated. The fluorescent-labeled RNC                 enhancing stability of medically relevant proteins in vivo.
was further confirmed by acidic SDS-PAGE with puromycine
treatment. From the time-resolved fluorescence lifetime                   We further used the system to assess the folding status of
measurements, the fluorescent donor lifetime was obtained                 intrinsically disordered proteins (IDPs) in presence or
and the FRET efficiency as well as donor-acceptor distance                absence of their binding partners in vivo. Co-expression of
was further calculated. Interestingly, our results suggested that         the right partner protein significantly decreases the proteolytic
the poly-alanine fragments were highly compacted within the               sensitivity of the IDP, indicating that the IDP becomes
tunnel universally. Considering the geometry of eukaryotic                more structured. We therefore provide a tool to address the
and prokaryotic ribosome exit tunnel in the literatures, we               unresolved question of if IPDs are also unstructured in the
speculated the nascent polypeptide could adopt secondary                  crowded environment in the cell.


181                                                                      salivary lipocalin with a different strand order in its β-barrel:
                                                                         the positions of the second and third strands (B and C) are
Kinship of n→π* interactions and hydrogen bonds                          switched, generating a mixed parallel- antiparallel structure.
Amit Choudhary1, Ronald T. Raines2,3                                     Triabin has no direct sequence similarity to any characterized
  Graduate Program in Biophyics, University of Wisconsin-                canonical lipocalin. Instead, at least three hypothetical insect
Madison, 1525 Linden Drive, Madison, WI, 53706,                          lipocalin sequences have significant sequence similarity to
  Department of Biochemistry, University of Wisconsin-                   both triabin and nitrophorin 2, and thus link them through
Madison, 433 Babcock Drive, Madison, WI, 53706,                          transitive homology. To gain insight into the relationship
  Department of Chemistry, University of Wisconsin-Madison,              between sequence differences and structural rearrangements
1101 University Ave, Madison, WI, 53706                                  in the salivary lipocalins, we structurally characterized one of
Noncovalent interactions modulate biomolecular structure,                these intermediate sequences, triabin-like lipocalin 4 (TLL4).
function, and dynamics. Electron delocalization is a                     Using solution NMR data, including backbone chemical shift
fundamental feature of many noncovalent interactions. For                assignments and long-range noe correlations, we established
example, a hydrogen bond involves delocalization of the lone             that TLL4 has a canonical lipocalin strand order. This is a
pair (n) of the hydrogen bond acceptor over the antibonding              surprising result given TLL4’s higher sequence similarity to
orbital (σ*) of the hydrogen bond donor. We discovered an                triabin than to nitrophorin 2 in the strand-swapped region of
interaction in proteins, termed the n→π* interaction, with               the barrel. Mapping of strand location and register onto a
similar electron delocalization. In this interaction, the lone           sequence alignment of TLL4 and triabin also revealed a five-
pairs (n) of an oxygen (Oi-1) of a peptide bond overlap with             residue register shift for the two swapped strands. The high
the antibonding orbital (π*) of Ci=Oi of the subsequent peptide          sequence similarity between TLL4 and triabin suggests that a
bond. Just as was the case with hydrogen bonds, this intimate            few critical sequence differences may govern strand swapping;
interaction between adjacent carbonyl groups was thought                 the observation of a strand register shift also suggests that the
to be of primarily electrostatic origin. By installing isosteric         structural rearrangement is more complicated than a simple
chemical substituents in a peptidic model system, and using              strand swap.
NMR spectroscopy, X–ray diffraction analysis, and ab
initio calculations to analyze the consequences, the intimate
interaction between adjacent carbonyl groups was shown                   Protein Networks (183)
to arise primarily from n→π* electronic delocalization. A
signature of this interaction is a short contact between the             183
carbonyl groups. The absence of n→π*interaction prevents
                                                                         A computational protocol for quantifying interaction
the development of these short contacts. An extensive survey
                                                                         specificity between protein families
of high resolution structures in the protein data bank suggested
                                                                         Loretta Au1, David Green1,2
widespread occurrence of such short contacts. Evaluation of              1
                                                                          Applied Mathematics and Statistics, 2Graduate Program in
these short contacts using ab initio computational analysis
                                                                         Biochemistry and Structural Biology, Stony Brook University,
indicated energetically meaningful n→π*interactions in
                                                                         Math Tower, Stony Brook, NY, 11794-3600
several fundamental structural elements in proteins, including
α-helices, 310 helices, and polyproline type-II helices (PPII).          Many cellular phenomena result from interactions between
Another signature of this interaction, the pyramidalization of           members of large protein families; while the interactions
the carbonyl carbon, was observed in the atomic resolution               made by distinct members of a family share many features, it
crystal structures of α-helical and 310 helical peptides and             is their differences that results in diverse biological activities.
proteins. These findings have important implications for                 An understanding of the underlying rules of how specific
protein structure, folding, and function.                                protein interactions are made would significantly enhance
                                                                         our ability to transfer insights between related systems, but
This work was supported by grant R01 AR044276 (NIH).
                                                                         remains an open problem. Here, we present work towards
                                                                         a novel computational protocol to directly address these
182                                                                      questions. We have selected heterotrimeric G-proteins as
Two salivary lipocalins from blood-sucking insects have                  a model system: this class of proteins is critical in various
different folds despite significant sequence similarity                  signal transduction pathways; experimental studies have
Christian Roessler, Matthew H. J. Cordes                                 shown interesting patterns of binding specificity between
Chemistry and Biochemistry, University of Arizona, 1041 E                subunits; and a wealth of structural, biochemical and cell
Lowell St., BSW, Rm. 436, Tucson, AZ, 85721                              biological data exists to be leveraged. Our protocol focuses
                                                                         on defining the entire sequence space that is compatible with
The salivary proteome of blood-sucking insects is comprised
                                                                         the formation of a given complex, using search algorithms
almost exclusively of anti-hemostatic lipocalins. Members
                                                                         derived from existing techniques in computational protein
such as the nitrophorins adopt a common eight-stranded anti-
                                                                         design. These algorithms allow us to efficiently conduct in
parallel β-barrel structure. Triabin is a structurally divergent


silico mutagenesis over an immense combinatorial search                   protein has a single DNA-binding site, which is located
problem. A hierarchical approach grounded in statistical                  centrally within the dimer and has a functionally homogeneous
analysis is used to provide guarantees on the completeness                structure. The stoichiometry and photocrosslinking data
of our conformational sampling, directing the refinement of               show that only a single monomer of the PriB dimer engages
the solution space at each successive step. The information               in interactions with the nucleic acid. The analysis of the
derived from our guided search can then be used to construct              PriB binding to long oligomers was done using a statistical
a network that illustrates the relationships between different            thermodynamic model that takes into account the overlap of
sequences in a visually meaningful way. This quantification               potential binding sites and cooperative interactions. The PriB
offers a new perspective that improves our conceptualization              dimer binds the ssDNA with strong positive cooperativity.
of how sets of related proteins may interact.                             Both the intrinsic affinity and cooperative interactions are
                                                                          accompanied by a net ion release, with anions participating
                                                                          in the ion exchange process. The intrinsic binding process
Protein-Nucleic Acid Interactions                                         is an entropy-driven reaction, suggesting strongly that the
(184 – 191)                                                               DNA association induced a large conformational change in
                                                                          the protein. The PriB protein shows a dramatically strong
                                                                          preference for the homo-pyrimidine oligomers with an
184                                                                       intrinsic affinity higher by about three orders of magnitude,
Metal Selectivity in the Ferric Uptake Regulator                          as compared to the homo-purine oligomers. The significance
Michelle Dominguez, Stephen Mills                                         of these results for PriB protein activity is discussed.
Chemistry and Biochemistry, University of San Diego, 5998
Alcala Park, San Diego, CA, 92110                                         186
This research is focused on understanding how metal                       Kinetics of protein translocation between two DNA
selectivity is produced in the Ferric Uptake Regulator (Fur).             duplexes at equilibrium: Validation of NMR studies
Fur is a repressor protein found in bacteria cells that, when             Debashish Sahu, Junji Iwahara
activated by iron, binds to DNA and blocks transcription of               Biochemistry and Molecular Biology, The University of Texas
genes that promote iron intake. We have cloned and expressed              Medical Branch at Galveston, 301 University Boulevard,
Fur from several organisms, including Shewanella oneidensis               Galveston, TX, 77555-0647
and Pseudomonas aeruginosa. Using metal titration methods,
the affinities of the different homologues were measured for              Nonspecific DNA interactions are considered to play a crucial
various metals, including Co(II), Mn(II), Zn(II), and Fe(II).             role in increasing the efficiency with which a DNA-binding
Using surface binding assays, we determined the affinity of               protein locates its specific target site. In the target search
each Fur homolog for DNA with different metals bound.                     process, proteins change their locations via mechanisms such
These affinities, in conjunction with the sequences of the                as sliding and direct transfer. Recently some NMR approaches
proteins, will be used to identify the amino acids that may be            that permit the kinetic investigation of this dynamic process
important for metal selectivity in Fur.                                   at equilibrium have been proposed. Here we present the
                                                                          theoretical validation of the NMR methods. Applying the
                                                                          Kramer’s theory on kinetics of stochastic processes, we
185                                                                       considered the kinetics of intermolecular translocation of
Interactions of the E. coli Primosomal PriB Protein with                  proteins between two DNA molecules at equilibrium. The use
the Single-stranded DNA. Solution vs. Crystallographic                    of the Kramer’s theory permits kinetic simulations without
Studies.                                                                  breaking the rule of detailed balance for a general N-state
Michal Szymanski, Maria Jezewska, Wlodek Bujalowski                       model. The rate constants for sliding, dissociation, and direct
Biochemistry and Molecular Biology, University of                         transfer were considered as functions of free energies for
Texas Medical Branch at Galveston, 301 University Blvd,                   microscopic states and transitions states. Based on the kinetic
Galveston, TX, 77555                                                      theory together with the McConnell equations, we simulated
Quantitative analysis of the interactions of the Escherichia coli         NMR data for a system where a protein moves from one
primosomal PriB protein with a single-stranded DNA was done               DNA site to another via 1) sliding, 2) dissociation & re-
using quantitative fluorescence titration, photocrosslinking, and         association, and 3) direct transfer. Obtained results indicate
analytical ultracentrifugation techniques. Stoichiometry studies          that the previously proposed NMR approaches accurately
were done with a series of etheno-derivatives of single-stranded          provide kinetic rate constants for the intermolecular protein
(ss) DNA oligomers. Interactions with the unmodified nucleic              translocations between two DNA molecules, if sliding is faster
acids were studied, using the macromolecular competition                  than the intermolecular protein translocation. Qualitative
titration (MCT) method. The total site-size of the PriB dimer–            information on the timescale of the sliding process can also
ssDNA complex, i.e. the maximum number of nucleotides                     be obtained if the direct transfer mechanism is predominant
occluded by the PriB dimer in the complex, is 12 ± 1 nt. The              in the intermolecular protein translocation.


187                                                                    188
The N-terminal domain of murine hepatitis virus                        Role of a Novel Domain in Splicing Factor 1 during the
nucleocapsid protein binds specifically to the                         Early Stages of Pre-mRNA Splicing
transcriptional regulatory sequence and plays a critical               Ankit Gupta1, Clara L. Kielkopf2
role in viral replication                                              1
                                                                        Department of Biochemistry and Biophysics, University
Sarah Keane1, Pinghua Liu2, Julian Leibowitz2, David                   of Rochester, 601 Elmwood Avenue, Box 712, Rochester,
Giedroc3                                                               NY, 14642, 2Department of Biochemistry and Biophysics,
  Department of Chemistry, Indiana University, 800 E.                  University of Rochester, 601 Elmwood Ave, Box 712,
Kirkwood Ave, Bloomington, IN, 47405, 2Department of                   Rochester, NY, 14642
Microbial and Molecular Pathogenesis, Texas A&M Health                 Splicing Factor 1 (SF1) and U2 snRNP auxiliary factor
Science Center, College of Medicine, Texas A&M Health                  (U2AF65) form an essential protein complex that recognizes
Science Center College of Medicine, 407 Reynolds Medical               the 3’ splice-site during the initial stages of pre-mRNA
Building, College Station, TX, 77843, 3Chemistry, Indiana              splicing. A ~100 amino acid domain of SF1 (the ‘mystery’
University, 800 E. Kirkwood Ave, Department of Chemistry,              domain) is located between the N-terminal U2AF65-interacting
Bloomington, IN, 47405                                                 region, and a C-terminal RNA-binding domain. Despite high
Coronavirus (CoV) nucleocapsid (N) protein is an RNA                   sequence conservation from yeast to mammals, the structure
chaperone that packages genomic RNA and has been                       and function of this SF1 mystery domain is currently
implicated in viral replication and sub genomic (sg) RNA               unknown. Here, we demonstrate that the SF1 mystery
synthesis. N is a multi-domain protein that contains two               domain participates in the SF1/U2AF65 interface and splice-
structurally independent RNA binding domains denoted                   site recognition by comparing changes in heat capacities,
N-terminal domain (NTD) and C-terminal domain (CTD).                   chemical shifts, and affinities for U2AF65 complexes with
These domains are joined by a charged linker region that               deletion variants of SF1.
is rich in serine and arginine residues (SR-linker) that is            Heat capacity changes for association of SF1 with the U2AF65-
likely a target for reversible phosphorylation in the cell.            interacting domain (UHM) are significantly greater than
The NTD forms a sequence specific high affinity complex                those observed with the minimal U2AF65-interacting peptide
with the transcriptional regulatory sequence (TRS) RNA, a              in SF1. Since heat capacity changes often correlate with the
conserved hexanucleotide sequence that is critical for viral           amount of surface area buried by complex formation, it is
replication while the CTD mediates the dimerization of N               likely that additional regions of SF1 participate in the U2AF65-
and likely binds RNA non-specifically via electrostatic                UHM interface. To investigate this possibility, we compared
interactions. Murine hepatitis virus (MHV) NTD (residues               the HSQC spectra of 15N-labeled U2AF65-UHM in complex
64-194) adopts a U-shaped β-platform containing five                   with SF1 deletion variants. Chemical shift differences imply
short β-strands (arranged β4-β2-β3-β1-β5) and an extended              that the SF1 mystery domain participates in the U2AF65-
β-hairpin, a fold that is almost identical to the NTDs of              UHM interface. Furthermore, isothermal titration calorimetry
other CoV N proteins. The putative RNA binding groove is               (ITC) experiments establish a three-fold enhancement in the
characterized by a large patch of basic and aromatic amino             affinity between SF1 and U2AF65-UHM in the presence of
acids across the β-platform that are proposed to directly              SF1 mystery domain compared to the minimal N-terminal
interact with RNA. Fluorescence anisotropy-based RNA                   interaction region.
binding experiments with 5’-fluorescein-labeled TRS and
MHV NTD reveal that Y105A, H107A and H136A mutant                      Complementary fluorescence anisotropy assays and ITC
proteins each bind to the TRS RNA with near wild type                  experiments reveal that the mystery domain affects the
affinity; in contrast, the affinities of R125A and Y190A NTDs          affinity of SF1/U2AF65 complex for pre-mRNA splice-sites.
are markedly decreased. Reverse genetic studies show that              From fluorescence anisotropy assays, a seven-fold reduction
viruses containing the Y105A, H107A and H136A mutations                in the affinity of the SF1/U2AF65 complex for the AdML
are all viable, however, the R125A mutation is lethal. These           splice-site was observed for the SF1Δmystery/U2AF65
results indicate that the “front” of the platform (nearest the         complex. Comparable differences in the affinities of the
SR-linker) are critical TRS binding determinants essential             two complexes were observed by ITC for an α-tropomyosin
for virus viability. These data are fully consistent with an           splice-site. We propose that SF1 mystery domain is important
NTD chemical shift perturbation map obtained upon TRS                  for SF1/U2AF65/RNA assembly in the early stages of pre-
binding. The effect of these mutations on the structure and            mRNA splicing.
RNA-binding properties of full-length N are currently under            Funding support from: NIGMS (2R01GM070503-07)
This project is funded by R01 AI067416 (to D. P. G. and J. L.
L.) from the NIH.


189                                                                       190
Nucleotide analogs and molecular modeling studies reveal                  Streptococcus pneumoniae NmlR is a single cysteine
key interactions involved in substrate recognition by the                 containing nitric oxide sensor that forms a stable S-NO
yeast RNA triphosphatase.                                                 adduct
Moheshwarnath Issur1, Simon Despins2, Isabelle Bougie2,                   Gregory Campanello, David Giedroc
Martin Bisaillon2                                                         Department of Chemistry, Indiana University, 800 E.
  Biochemistry, Université de Sherbrooke, 3001, 12e Avenue                Kirkwood, Bloomington, IN, 47405
Nord, Sherbrooke, QC, J1H5N4, Canada, 2Biochemistry,                      S. pneumoniae Neiserria MerR-like regulator (NmlR)
Université de Sherbrooke, 3001, 12e Avenue Nord,                          is a proposed nitric oxide (NO) sensor that regulates
Sherbrooke, QC, J1H5N4, Canada                                            the expression of an S-nitrosylated glutathione (GSNO)
Objective: RNA triphosphatases (RTPases) are involved                     reductase, adhC. NmlR and AdhC are thought to work in
in the addition of a distinctive cap structure found at the 5’            concert to comprise an NO detoxification system at the site
ends of eukaryotic mRNAs. Fungi, protozoa and some DNA                    of a pneumococcal infection. NmlR is postulated to use a
viruses possess an RTPase that belongs to the triphosphate                single conserved cysteine, C52, to form an S-NO bond
tunnel metalloenzyme family of enzymes that can also                      followed by transcriptional activation via a DNA distortive,
hydrolyze nucleoside triphosphates. Previous crystallization              MerR-like mechanism. ESI-MS and UV-Vis absorbance
studies revealed that the phosphohydrolase catalytic core                 experiments indicate that NmlR forms a stable S-NO adduct
is located in a hydrophilic tunnel composed of antiparallel               in vitro when reacted with GSNO, and that this adduct is
beta-strands, without however providing any information on                protected from reduction by an S-NO bond specific reducing
the contacts between the enzyme and the triphosphate, sugar               reagent, ascorbate. C52 is also reactive with other NO
or base moieties of the phosphohydrolase substrate. This is               donors, S-methylating reagents and hydrogen peroxide.
not surprising since thermodynamic studies have shown that                Fluorescence-based DNA binding experiments have shown
the binding of the RNA or nucleotide substrates to RTPases                that a C52A mutant binds a 20 base-pair operator containing
results in a destabilization of the enzymes. Methods: In the              DNA with 19-fold weaker affinity than WT NmlR. This
present study, we used computational molecular docking to                 suggests a coupling between C52 and the DNA binding region
model the binding of a nucleotide substrate into the yeast                of NmlR. Taken together, the data suggest C52 is initially
RTPase active site. In order to confirm the docking model and             exposed and highly reactive. Upon S-NO derivatization,
to gain additional insights into the molecular determinants               a conformational change is hypothesized to protect C52
involved in substrate recognition, we also evaluated both                 which also results in transcriptional activation. Results from
the phosphohydrolysis and the inhibitory potential of an                  ongoing studies focus on the mechanism of transcriptional
important number of nucleotide analogs. Results: Our study                regulation by NmlR and an investigation of the chemical
highlights the importance of specific amino acids for the                 environment around C52 using fluorescence and NMR based
binding of the sugar, base and triphosphate moieties of the               studies will be presented.
nucleotide substrate. In addition, unnatural substrates such as           Supported by a grant from the National Institutes of Health
nucleotide analogs or tripolyphosphate can still be hydrolyzed            (R01 GM042569)
even if the conformation of the active site is less than optimal,
lacking several important contacts with the substrate. We also            191
identified some nucleotide analogs which could bind tightly
to the yeast RTPase without being efficiently hydrolyzed.                 Structural Flexibility of the Cytidine Repressor DNA-
Conclusion: Our structural docking model, coupled with the                Binding Domain
use of an important number of nucleotide analogs, highlights              Colleen Moody1, Vira Tretyachenko-Ladokhina2, Donald F
the importance of specific residues for the binding of the                Senear3, Melanie J Cocco4
nucleotide substrate, and reveals both the structural flexibility         1
                                                                           Molecular Biology and Biochemistry, University of
and complexity of the active site. Our study illustrates the              California, Irvine, 1413 Natural Sciences I, Irvine, CA,
important structural and functional features for the interaction          92697, 2Molecular Biology and Biochemistry, University
of an RTPase with a ligand and opens the way to the use of                of California, Irvine, 3305 McGaugh Hall, Irvine, CA,
nucleotide analogs as potential inhibitors of the RTPases of              92697, 3Molecular Biology and Biochemistry, University
pathogens.                                                                of California, Irvine, 3230 McGaugh Hall, Irvine, CA,
                                                                          92697, 4Molecular Biology and Biochemistry, University
                                                                          of California, Irvine, 1218 Natural Sciences I, Irvine, CA,
                                                                          The structure of the Cytidine Repressor (CytR) DNA-
                                                                          binding domain (DBD) in the presence of a recognition site
                                                                          oligonucleotide has been solved using Nuclear Magnetic


Resonance (NMR). The structure reveals a helix-turn-                      hydrophobic residues in holo-S100A13, which is a unique
helix motif common to DNA-binding proteins. However,                      characteristic of S100A13. 1H-15N- HSQC NMR experiments
the structural stability of the domain depends on the DNA                 reveal the binding site of the Annexin II peptide on holo-
sequence with which it interacts. Hydrogen-deuterium                      S100A13 to be distinctly different from other S100/Annexin
exchange and temperature induced unfolding were used to                   interactions. In order to define the specificity of S100A13
assess the structural stability of the DBD in three conditions.           for Annexin II specifically, the interaction of S100A13 with
With no DNA present, the CytR DBD has greater overall                     other Annexin peptides was characterized by ITC. Site-
flexibility and adopts two conformations in slow exchange                 Directed mutagenesis was used to mutate key residues in the
on the NMR timescale. In the presence of a nonspecific DNA                Annexin II binding interface on S100A13. The information
sequence, the DBD folds to a single structure, but still retains          gained from this study provides valuable information on the
the greater flexibility seen with the free protein. The structure         interaction between S100A13 and the Annexin II peptide and
solved in the presence of recognition site DNA has the                    gives much needed insight into the mysterious pathway of
highest thermal stability as well as protection from exchange.            non-classical release of the signal peptide-less proteins.
We conclude that the CytR DBD is a dynamic domain
that is strongly influenced in stability and structure by the             193
environment created by the DNA sequence. Protein folding
coupled to DNA recognition is a contrast between CytR and                 Understanding the structure and stability of FGF-1 in a
the DBD of LacR. Thus, although CytR is a member of the                   cellular environment using NMR spectroscopy
LacR family, DNA recognition invokes unique properties.                   Daniel Vo1, T.K.S. Kumar2
This work was supported by the National Science Foundation
                                                                           University of Arkansas, 119 Chemistry Building, Fayetteville,
[MCB 02115769, MCB 06652875].                                             AR, 72701, 2Chemistry and Biochemistry, University of
                                                                          Arkansas, University of Arkansas, 119 Chemistry Building,
                                                                          Fayetteville, AR, 72701
Protein Structure/Function Studies                                        Much of the research on biological proteins is performed (in
(192 – 215)                                                               vitro) under artificial conditions involving the isolation of a
                                                                          protein from the cell. The living cell, however, constitutes
                                                                          a very complex system, and a protein’s native structure and
192                                                                       stability may be influenced by its environment. Thus, “in-cell”
Defining the Interaction between S100A13 and Annexin                      studies of a protein are critical to understanding the protein’s
II Peptide: Insight into Non-classical Secretion                          structure and stability under physiological conditions.
Anna Daily, Ashley Martfeld, T.K. Suresh Kumar                            The primary goal of the study is to examine the effects of
Chemistry and Biochemistry, University of Arkansas, 119                   the intracellular environment on the structure and stability
Chemistry Building, Fayetteville, AR, 72701                               of Fibroblast Growth Factor 1 (FGF-1) through the use of
S100A13 is a calcium binding chaperone protein that is                    Nuclear Magnetic Resonance (NMR) spectroscopy. Certain
known to be involved in the non-classical export of signal                limitations, however, are associated with in-cell NMR studies
peptide-less proteins, such as fibroblast growth factor                   (including the high concentration of isotope labeled protein
(FGF-1) and interleukin-1α. It has also been shown that the               required). In-cell conditions can be simulated (in vitro)
interaction of S100A13 with Annexin II, which exhibits an                 through the addition of bacterial intracellular proteins to a
inducible flip-flop mechanism across the cell bilayer, helps              NMR sample. In the experiment, FGF-1 was over expressed
the multiprotein release complex to traverse the membrane                 in recombinant Escherichia coli (E. coli), extracted by
bilayer. The interaction of S100A13 and Annexin 2 has been                sonication, and purified by column chromatography. FGF-
characterized using various biophysical techniques including              1 is a protein involved in cellular proliferation and wound
Isothermal Titration Calorimetry (ITC), Differential                      healing. Little information is known regarding FGF-1’s
Scanning Calorimetry (DSC), and multidimensional NMR                      interactions inside the cell, as it follows a non-classical
spectroscopy. Results of the Isothermal titration calorimetry             secretion pathway. To better understand the structure of FGF-
(ITC) experiments show that holo-S100A13 exhibits                         1 under physiological conditions, a series of HSQC “titration”
preferential binding to Annexin II with high affinity in the low          experiments were performed to monitor changes in the
micro molar range compared to apo-S100A13. Equilibrium                    backbone structure of 15N - labeled FGF-1 in the presence of
guanidine hydrochloride denaturation monitored by steady-                 bacterial proteins. The stability of the protein was determined
state fluorescence and limited trypsin digestion analysis both            by NMR spectroscopy through thermal denaturation studies.
reveal holo-S100A13 to be stabilized upon binding with the                Data analysis consisted of the resonance assignment of all
Annexin II peptide. ANS (8-anilino-1-napthalene sulfonate)                HSQC spectra and the construction of perturbation plots.
binding experiments indicate that the presence of Annexin                 The preliminary results of the study indicate that a moderate
II peptide does not increase the solvent accessiblity of                  number of amino acid residues are perturbed in the presence


of bacterial proteins. The data implies that protein structure            across the cell membrane by the membrane-bound protein
and stability are influenced by the in-cell environment.                  Annexin II. In order to further define the interactions in
Funding was provided through the University of Arkansas                   the multi-protein release complex of FGF-1, it is essential to
Honors College.                                                           create a monomer of S100A13. Site-directed mutagensis has
                                                                          been used to introduce mutations in the hydrophobic interface
194                                                                       of S100A13 creating a single mutant F73K and the double
                                                                          mutant F73K, L79E. Futhermore, a triple mutant F73K, L79E,
The Role of Heparin in Fibroblast Signaling                               L83Q will also be made. Introduction of F73K mutation
Rebecca Kerr, Nicole Webb, T.K.S. Kumar                                   shows significant destabilization of the protein by protelytic
Univeristy of Arkansas, 119 Chemistry Building, Fayetteville,             trypsin digestion. Data on the biophysical characterization
AR, 72701                                                                 of all mutations by ITC, DSC, Fluorescence, and multi-
Human acidic fibroblast growth factor (hFGF-1) belongs to                 dimensional NMR Spectroscopy will be presented.
a superfamily of 22 fibroblast growth factors (FGFs) that
have a high affinity to heparin. It is a 16kD protein, which is           196
involved in many cellular functions, such as, cell proliferation,         Bioinformatics and kinetic studies in human pyridoxal
cell differentiation, angiogenesis, tumor growth, and wound               kinase reveal a new motif associated to zinc coordination
healing. FGF-1 exhibits its cell proliferation activity by                and regulation of enzyme activity
binding to cell-surface tyrosine kinase receptors (FGFRs).                Victoria Guixé, César Ramírez
The binding of FGF-1 to FGFRs results in dimerization of                  Facultad de Ciencias, Universidad de Chile, Las Palmeras
the receptor, which in turn, leads to its activation. Heparin             3425, Santiago, Chile
is proposed to have two functions: it protects FGFs from
degradation by proteases and from thermal denaturation, and               Human pyridoxal kinase (hPLK) catalyses the ATP-
it creates a local extracellular reservoir of FGFs, required              dependent phosphorylation of the 5’ hydroxymethyl group of
for receptor binding and activation. However, it remains                  pyridoxal in the presence of divalent metal cations, like zinc
undetermined whether or not heparin is required for both                  or magnesium. It belongs to the ribokinase superfamily, and
purposes. In this study, we attempt to understand the structural          possesses conserved tertiary structure and sequence motifs
role of heparin in the FGF signaling process. In this context,            common to all of its members. Pyridoxal quinase is the smallest
several mutations within the heparin-binding region of FGF-1              protein of this superfamily and along with hydroxymethyl
were constructed. Some of the mutants in the heparin-binding              pyrimidine (HMPK) and hydroxyethyl thiazole (THZK)
pocket completely knocked out the binding affinity of FGF to              probably represents the ancestral architecture.
the proteoglycan. The structural and functional consequences              Resolved structures of human and sheep pyridoxal kinase
of the mutations in the heparin-binding pocket have been                  have shown that Asp113 participates on the coordination
examined using a variety of biophysical techniques and cell               of divalent cations on the metal-nucleotide complex inside
proliferation assay.                                                      the active site. This interaction was also observed on crystal
                                                                          structures of THZK from B. subtilis and HMPK from S.
195                                                                       typhimurium.

Development of a S100A13 Monomer in Order to Further                      In order to determine if this aspartic acid is conserved between
Define the Non-classical Release Pathway of FGF-1                         PLK, HMPK and THZK, we studied the evolution of this
Ashley Martfeld1, Emily Erstine2, T.K.S. Kumar3, Anna                     protein family using the Bayesian method of phylogenetic
Daily3                                                                    inference and real value evolutionary trace. Our results
  Chemistry, University of Arkansas, 119 Chemistry Building,              indicate that the eukaryotic PLK group is more related with
fayetteville, AR, 72701, 2Chemistry, University of Arkansas,              the bacterial clade represented by the pdxy gene from E. coli,
119 Chemistry Building, Fayetteville, 0, 72701, 3Chemistry/               contrary to what was suggested before. Also, the aspartic
Biochemistry, University of Arkansas, 119 Chemistry                       residue is localized in a new 3-aminoacid motif (DPV) strictly
Building, Fayetteville, AR, 72701                                         conserved between the protein family.

Fibroblast growth factors are key proteins that play a crucial            We tested the role of the Asp113 from hPLK through site-
role in cellular processes such as angiogenesis, embryogenesis            directed mutagenesis and kinetic studies, using zinc as
and even tumor development. Fibroblast Growth Factor-1                    divalent cation. We determined that the wild-type enzyme
(FGF-1) is exported through a non-classical pathway as a part             was strongly inhibited by free zinc (IC50 of 75 ± 14 µM),
of a multi-protein complex with S100A13. The formation of                 while the D113N mutant was less inhibited (988 ± 137 µM).
this complex is driven by a Cu2+ dependent reaction that                  Surprisingly, the D113V mutant lost this behavior, now
binds an FGF-1 homo-dimer to the proteins p40 Syt1 and                    increasing its activity as a function of free zinc concentration,
S100A13. This multi-protein complex is then transported                   showing a half activator concentration (AC50) of 24 ± 5 µM.


These results suggest the importance of this new DPV motif                 198
in catalysis and regulation of enzyme activity in this protein
family. (Fondecyt 1070111).                                                Contact Surface Mapping of LysRS in its Multi-functional
                                                                           States by Solution-phase Hydrogen/deuterium Exchange
197                                                                        FT-ICR Mass Spectrometry
                                                                           Hui-Min Zhang1, Min Guo2, Xiang-Lei Yang2, Paul
Structure of D-AKAP2-PKA RI isoform complex: Insights                      Schimmel2, Qian Zhang3, Mark R. Emmett1,3, Alan G.
into AKAP specificity and selectivity                                      Marshall1,3
Ganapathy Sarma1, Francis Kinderman1,2, Choel Kim1,3,                      1
                                                                            ICR, National High Magnetic Field Lab, 1800 E. Paul Dirac
Sventja von Daake1, Susan Taylor1,4                                        Dr, Tallahassee, FL, 32310, 2The Scripps Research Institute,
  Chemistry and Biochemistry, University of California, San                10550 North Torrey Pines Road, La Jolla, CA, 92037,
Diego, 9500 Gilman Drive, Mail Code 0654, 432 Leichtag                     3
                                                                            Chemistry and Biochemistry, Florida State University, 95
Building, La Jolla, CA, 92093-0654, 2Amgen Inc., One Amgen                 Chieftain Way, Florida State University, Tallahassee, FL,
Center Drive, Thousand Oaks, CA, 91320, 3Pharmacology,                     32306
Baylor College of Medicine, One Baylor Plaza, Houston,                     In addition to aminoacylation, tRNA synthetases act in
TX, 77030, 4Howard Hughes Medical Institute, 9500 Gilman                   key signal transduction pathways. These novel functions
Drive, Mail Code 0654, 432 Leichtag Building, La Jolla, CA,                require conformation activation to redirect the synthetase
92093-0654                                                                 from translation to signal transduction. Not known are the
cAMP-dependent protein kinase (PKA) regulates a variety                    particulars of such changes. Here we use solution-based
of biological activities by phosphorylating a broad range                  hydrogen/deuterium exchange FT-ICR mass spectrometry
of target proteins. Spatial and temporal specificity of PKA                to reveal the conformational change associated with a novel
activity is, in turn, achieved by a family of scaffolding proteins         function of human lysyl-tRNA synthetase.
called as the A-kinase anchoring proteins (AKAPs). One of                  Mast cells are activated when an allergen binds to IgE-
these, dual-specific AKAP 2 (D-AKAP2) binds to both the                    FcεRI receptors. Upon immune activation, mammalian
Regulatory (R) isoforms (RI and RII) of PKA. Specifically,                 lysyl-tRNA synthetase (LysRS) is phosphorylated by ERK
the dimerization and docking (D/D) domain at the N-terminus                kinase and then translocates to the nucleus. There it binds to
of the R subunits forms a four-helix bundle that provides an               the Microphthalmia-associated transcription factor (MITF),
interface for AKAP binding.                                                leading to the activation of MITF-dependent gene expression
In order to understand the basis of D-AKAP2 binding to the                 [1]. In quiescent cells, LysRS resides in a large multi-
RIα subunit and understand the molecular determinants that                 synthetase complex (MSC) in the cytoplasm, together with
make D-AKAP2 dual-specific, we have solved the structures                  8 other tRNA synthetases and 3 scaffold proteins, including
of the RIα D/D domain with and without the D-AKAP2                         MSC p38 [2]. The binding site of LysRS with p38 is still
peptide. The structure of the apo crystal form was solved using            not known. Previous studies showed that phosphorylation
the anomalous signal from inherently present sulfur atoms.                 of Ser207 of LysRS can release the enzyme from the MSC
The structures highlight the importance of redox-sensitive                 and that a phosphorylation-mimicry mutation--S207D--
disulfides in affecting AKAP binding. An unexpected shift in               constitutively activates mast cells. We find that the S207D
the helical register of D-AKAP2 compared to RIIα:D-AKAP2                   mutation opens two domains in LysRS, and thus shifts the
complex structure makes the mode of binding to RIα, novel.                 dynamics of LysRS into a more flexible open state, where
Further, the comparison allows us to deduce a molecular                    it dissociates from the MSC (p38). The results also reveal
explanation for the sequence and spatial determinants of                   contact surfaces between LysRS and p38. These results reveal
AKAP specificity. This work was supported by HHMI,                         a dramatic flexibility of the tRNA synthetase architecture.
American Heart Association Postdoctoral Fellowship to                      Acknowledgements: NIH (GM 78359, AI33696), NSF
G.N.S, and NIH grants GM34921 and DK54441 to S.S.T.                        (DMR-0654118), and the State of Florida.
                                                                           1. Yannay-Cohen, N. et al. Mol. Cell, (2009), 34, 603-611
                                                                           2. Guo, M. et al. Proc. Natl. Acad. Sci. USA, (2008), 105(7),
                                                                           3. Zhang, H.-M. et al. Anal. Chem., (2008), 80 (23), 9034-


199                                                                      application is the L-lactate oxidase. A high selectivity of this
                                                                         enzyme is crucial for its efficient use.
Biochemical and Structural Studies of HMW1C-like
glycosyltransferases                                                     Several crystal structures of family members have been solved
Hye-Jeong Yeo, Kyoungjae Choi, Seonghee Paek, Fumihiro                   and allow detailed insight into the substrate binding site. We
Kawai                                                                    present a detailed analysis of the substrate binding sites of
Biology and Biochemistry, University of Houston, 4800                    selected members of this family and discuss the influence
Calhoun, Houston, TX, 77204                                              of certain residue on substrate selectivity. Variants of the
                                                                         enzymes prepared by site-directed mutagenesis with the aim
The Haemophilus influenzae HMW1 protein is a High-                       of changing the substrate selectivity will be presented.
Molecular Weight non-pilus adhesin that mediates attachment
to human epithelial cells, an essential step in the pathogenesis         201
of H. influenzae disease. The HMW1 adhesin is a prototype
exoprotein secreted via the two-partner secretion pathway and            High-resolution crystal structures of intact and cleaved
is glycosylated at multiple asparagine residues in consensus             forms of bovine pancreatic trypsin inhibitor (BPTI)
N-linked sequons. Contrast to the heavily branched glycans               variants bound to trypsin
found in eukaryotic N-linked secretory glycoproteins,                    Jonathan Penfield1, Elena Zacharova1, Scott Classen2,
the modifying glycan structures in HMW1 are simple                       Martin P. Horvath1, David P. Goldenberg1
mono-hexoses or di-hexoses. A recent study established                   1
                                                                          Biology, University of Utah, 257 South 1400 East, Salt
that H. influenzae HMW1C, a cytoplasmic protein, is the                  Lake City, UT, 84112-0840, 2Physical Biosciences Division,
glycosyltransferase responsible for modifying HMW1 and                   Lawrence Berkeley National Laboratory, One Cyclotron
transfers glucose and galactose to acceptor sites. To understand         Road Mailstop: 6R2100, Berkeley, CA, 94720-8226
the molecular basis for the carbohydrate modification of                 In order to explore the relationships among structure, dynamics
HMW1, we have investigated the structure and function of                 and function in serine proteases and their natural inhibitors,
HMW1C-like proteins. An Actinobacillus pleuropneumoniae                  we are determining high-resolution crystal structures of
protein designated ApHMW1C shares high-level homology                    BPTI variants bound to anionic rat trypsin. For each BPTI
with HMW1C and has been assigned to the GT41 family,                     variant (wild-type, Y35G, G36N and G37A), three complex
which otherwise contains only O-glycosyltransferases.                    types were formed and crystallized: the intact inhibitor with
Here we show that ApHMW1C is an N-glycosyltransferase.                   active trypsin; the intact inhibitor with an inactive trypsin
ApHMW1C was able to transfer glucose and galactose to                    variant (S195A); and the cleaved inhibitor (with the Lys15-
known asparagine glycosylation sites in HMW1, as assessed                Ala16 scissile bond hydrolyzed) bound to S195A trypsin.
by fluorescent glycoprotein detection methods in vitro. Initial          The structures containing the cleaved inhibitors represent
structure-function studies suggested that ApHMW1C has a                  enzyme-product complexes in which the enzyme is poised to
15-kDa N-terminal domain and a 55-kDa C-terminal domain                  resynthesize the peptide bond. Structures of all 12 complexes
that harbors glycosyltransferase activity. These findings                have been determined and refined, in some cases against
establish a new family of N-glycosyltransferases distinct                x-ray data with resolution limits better than 1.1 Å.
from GT41 family O-glycosyltransferases. Furthermore,
our progress on the crystal structure of ApHMW1C will be                 Comparisons of these structures indicate that the various
presented and discussed.                                                 substitutions, as well as hydrolysis of the peptide bond,
                                                                         are accommodated with minimal perturbation of the native
This work was supported by the Robert A. Welch Foundation                structure. There are, however, significant differences among
grant E-1616 and NIH grant AI068943 to H.J.Y.                            the temperature factors for the various structures. In all cases,
                                                                         the B-values for the inhibitor backbone display a pattern in
200                                                                      which the largest values are observed for the residues most
Structure-function relationships for substrate selectivity               distant from the trypsin-binding region. This pattern is not
in 2-hydroxy acid oxidizing enzymes                                      observed in structures of the free inhibitor and are suggestive
Stefan Leitgeb1,2, Thomas Stoisser1,2, Daniela Neuhold1,2,               of a bending motion within the inhibitor, centered about a
Bernd Nidetzky2                                                          hinge located relatively close to the binding site. Anisotropic
  Research Center Pharmaceutical Engineering GmbH,                       temperature factors calculated for the highest-resolution
Inffeldgasse 21a/II, Graz, 8010, Austria, 2Institute of                  structures provide further support for this interpretation. The
Biotechnology and Biochemical Engineering, Graz University               B-values for residues distal from the binding site appear to
of Technology, Petersgasse 12/1, Graz, 8010, Austria                     be enhanced in the complexes containing the cleaved mutant
                                                                         inhibitors. Further refinement and analysis of these structures
The substrate spectrum for members of the family of α-hydroxy            is in progress and is expected to provide new insights into
acid oxidizing enzymes ranges from short substrates like                 the mechanism of inhibition by BPTI and other standard-
glycolate (C2) to L-2-hydroxypalmitate (C16). The most                   mechanism inhibitors.
prominent member of this family in terms of industrial


202                                                                        sequence that may form a membrane-anchoring helix. The
                                                                           central B domain (residues 73-195) has been described as
Systematic Approach to Better Crystals                                     pore-forming. It shares sequence homology with the “BON”
Qiang Zhao, Qian Wang, Siwei Chen, Tiantang Dong, Xinjun                   domain of Bacterial Osmotic-shock-resistance, Nodulation-
Liu, Xiaoshu Hou, Yuanna Zhai, Shourong Liu, Yan Liu,                      specificity and lipid-binding proteins. The C domain (residue
Fangfang Xiao, Yujun Han, Huili Hou, Ming Liu, Chong Qi,                   212-326) shares significance homology with other bacterial
Yike Wang, Qiujing Huang, Chen Ke, Yang Sun, Qi Wang,                      peptidoglycan-binding domains, including the C-terminus
Michelle Yu Xia, Zhongmin Maxwell Wang                                     of the E. coli outer membrane protein OmpA, after which
Structural Biology, Crown Bioscience, Inc., No.21 Huoju                    Rv0899 was originally named. Using NMR spectroscopy,
Street, Changping District, Beijing, China                                 we show that residues 73-326, spanning the joint B and C
Crystallography and structure-based drug design have                       domains, adopt a well-defined three-dimensional structure
become an integral part of the drug discovery process. The                 in water solution, however, the individual B and C domains
critical step is to obtain high purity protein to get diffraction-         fold independently. The B domain adopts a previously
quality crystals for x-ray data collection. While screening                undocumented fold, with three parallel/antiparallel α-helices
large number of conditions is usually the typical approach,                packed against six parallel/antiparallel β-strands which form
the process of crystallization remains impossible to predict.              a flat β-sheet. The core of the B domain is hydrophobic while
At Crown Bio., we have implemented a systematic process                    the exterior is polar and predominantly acidic. The relative
XtalMax™ involving multiple expression systems combined                    orientation of domains B and C, and the overall architecture of
with careful characterizations and post-translational                      the protein suggest alternative possible modes of membrane
treatments, which greatly increases the success rates for                  association.
diffraction-quality crystals.
1. Vigilant construct design: Use sequence and structure                   204
   alignment to determine cloning boundaries and purification              Calmodulin activation of estrogen receptor alpha
   tags, and to employ surface mutations to increase potential             Ramona Bieber Urbauer1, Carrie Jolly1, Savannah Johnson1,
   intermolecular interactions.                                            John Galdo1, Marie Cross1, Madeline Elliott1, Michael
2. Multiple expression systems: Enhance protein express                    Nooromid1, Leah Cho1, Noelle Cheung1, Brian Jones2, Erik
3. Co-expression: Produce homogeneous               protein    in          Henderson1, Jeffrey Urbauer1
   phosphorylation and glycosylation.
                                                                             Chemistry / Biochem. and Mol. Biol., The University of
                                                                           Georgia, Davison Life Sciences Complex, 120 E. Green St.,
4. Post-translational  treatments:  Remove      unwanted
                                                                           Athens, GA, 30602, 2Bruker AXS, Inc., 5465 East Cheryl
   phosphorylation and glycosylation with enzymatic
                                                                           Parkway, Madison, WI, 53711
   treatment, and reduce molecular flexibility with lysine
   methylation.                                                            The objective is to ascertain the physical underpinnings of
                                                                           calcium-dependent estrogen receptor alpha (ERa) activation
With carefully produced protein and high throughput
                                                                           by calmodulin (CaM) and how oxidative stress might mediate
screening of our proprietary screens using Mosquito™ robot,
                                                                           the interactions of CaM with ERa and antiestrogens. ERa
we considerably improved the chance of getting crystals with
                                                                           is the principal target for systemic endocrine/antiestrogen
very minimal amount of protein.
                                                                           therapy for estrogen-dependent breast cancers. The calcium
                                                                           signaling protein CaM promotes calcium-dependent ERa
203                                                                        activation and is an obligate activator. Antiestrogens such
Structure of the Mycobacterium tuberculosis virulence                      as tamoxifen and its metabolites bind tightly to CaM, and it
factor Rv0899(OmpATb)                                                      has been suggested that therapeutic benefit of antiestrogens
Yong Yao1, Peter Teriete1, Adrian Kolodzik1, Jinghua Yu1,                  for estrogen-dependent breast cancers may derive partially
Houhui Song2, Michael Niederweis2, Francesca Marassi1                      from CaM antagonism. We have localized the CaM binding
  Sanford Burnham Medical Research Institute, 10901                        region of ERa and initiated structural studies to determine the
North Torrey Pines Road, La Jolla, CA, 92037, 2University                  solution structure of the complex of CaM with the ERa CaM
of Alabama at Birmingham, University of Alabama at                         binding region. NMR chemical shift changes and structural
Birmingham, Birmingham, AL, 35292                                          models from small angle X-ray scattering (SAXS) indicate
                                                                           that CaM bound to ERa is somewhat extended structurally
Rv0899 (OmpATb) is a 326-residue membrane-associated
                                                                           compared to high affinity CaM complexes. The ERa CaM
virulence factor of Mycobacterium tuberculosis (Mtb),
                                                                           binding region adopts partial helical character upon CaM
the causative agent of tuberculosis. It is essential for the
                                                                           binding, suggesting that activation involves helix stabilization.
adaptation of Mtb to acidic environments and has been
                                                                           Binding of CaM to hydrophobic antiestrogens such as
identified as an outer membrane protein. The N-terminal
                                                                           tamoxifen, its metabolites (4-hydroxytamoxifen, endoxifen)
domain (residues 1-72) contains a 20-residue hydrophobic
                                                                           and raloxifene is abrogated by oxidation of the methionine


residues in CaM, but oxidation does not eliminate binding                   (HSA) were analyzed by circular dichroism and acrylamide
to ERa. Control experiments with CaM where methionine                       quenching whereas for global changes dynamic light
residues are replaced with leucine indicate that methionine                 scattering was performed. The alteration in conformational
residues are not essential for hydrophobic drug binding. The                stability (Tm from 65 to 73°C) of protein was explained on
results are important for a comprehensive understanding of                  the basis of helical content induction (from 55 to 75 %)
CaM activation of ERa and the link between oxidative stress                 when protein molecules come in to the contact of pollutants.
and development of antiestrogen resistance. The research                    The flexibility or constraint among the three domains was
was supported by the DOD, the Georgia Cancer Coalition,                     also explained with the help of other previously reported
Bruker AXS and the University of Georgia.                                   results on temperature mediated albumin unfolding. To
                                                                            authenticate the trustworthiness of the above explanations
205                                                                         for conformational changes, a correlation with functional
                                                                            aspects of albumin β-hydrolase activity was taken into
Molecular determinants of Human Beta-defensin 3                             consideration by colorimetry. We found that though HSA is a
binding to Melanocortin Receptors 1 and 4                                   monomeric protein but it shows the heterotropic allostericity
Matthew Nix1, Chris Kaelin2, Tina Ta1, Greg Barsh2, Glenn                   for its β-lactamase activity in the presence pollutants, which
Millhauser1                                                                 act as K- and V-type non-essential activator. Pollutants
  Department of Chemistry, University of California at Santa                cause conformational changes and bring about catalytic
Cruz, 1156 High St, Santa Cruz, CA, 95064, 2School of                       modification to the protein (from 100 to 200%). In other words
Medicine - Department of Genetics, Stanford University,                     this protein exists in different conformations with different
Stanford Medical Center, 279 Campus Dr., Stanford, CA,                      catalytic properties determined by binding of an activator.
94305                                                                       This enhancement is taken to mean as due to deliverance
The Beta-defensins are a class of small, cationic proteins found            of structural itinerancy which is associated with catalytic
ubiquitously in a diverse array of organisms ranging from                   activity of an unstructured protein. This is the first report of
mammals to plants and insects. First recognized for their broad             its kind which not only elucidates the catalytic allostericity
antimicrobial activity, they have also been shown to function               of serum albumin through a mechanistic approach but also
in the adaptive mammalian immune system. More recently, we                  correlating this with non-microbial drug resistance as albumin
have discovered yet another role for defensins, as a new class              has property of self-hydrolysis of β-lactam drugs which is
of ligands for the melanocortin system. The melanocortin                    further upregulated by these pollutants due to induction of
system comprises a group of receptors and ligands known                     ‘open to close transition’ in the albumin.
to modulate physiological processes such as pigmentation
(MC1R) and energy regulation (MC4R) in vertebrates. Here                    207
we report results of a structure-function analysis of Human
Beta-defensin 3 (HBD3) in order to determine the critical                   The bifunctional roles of nickel ions in Helicobacter pylori
features of this protein that are responsible for its high affinity         [NiFe]-hydrogenase maturation pathway
binding to the melanocortin receptors 1 and 4. Our results                  Wei Xia1, Hongyan Li2, Kong-Hung Sze3, Hongzhe Sun2
indicate that binding is facilitated in an electrostatic manner
                                                                             Chemistry, The University of Hong Kong, Room 309
between positive charges on HBD3 and negative charges found                 ChongYuetMing Chemistry Building, The University of
on the extracellular portion of the melanocortin receptors.                 Hong Kong, Hong Kong, Hong Kong, Hong Kong (CN),
These findings identify a mode of melanocortin receptor-
                                                                             Chemistry, The University of Hong Kong, Room 503
ligand interactions distinct from previously studied ligands,               ChongYuetMing Chemistry Building, The University of
which further establishes the link between the immune and                   Hong Kong, Hong Kong, Hong Kong, Hong Kong (CN),
melanocortin system and has potential implications for ligand
                                                                             Chemistry, The University of Hong Kong, Room 603
design targeting the MC receptors.                                          ChongYuetMing Chemistry Building, The University of
                                                                            Hong Kong, Hong Kong, Hong Kong, Hong Kong (CN)
206                                                                         [NiFe]-hydrogenases are widely produced by many types
                                                                            of bacteria and involved in hydrogen metabolism. The large
Mechanism of pollutant-induced conformational                               subunit of the hydrogenase contains a dinuclear [NiFe] metal
alterations in human serum albumin: a key to catalytic                      center, which is required for enzyme activity. The insertion of
heterotropic allostericity causing non-microbial drug                       nickel ion into the metalocenter requires the participation of
resistance                                                                  two chaperones, HypA and HypB, a metal-binding GTPase.
Ejaz Ahmad, Gulam Rabbani, Rizwan Hasan Khan                                Although extensive studies have been performed, the detailed
Interdisciplinary Biotechnology Unit, Aligarh Muslim                        mechanism of nickel insertion carried out by these two
University, ., Aligarh, 202002, India                                       proteins remains unknown.
The pollutant (1-naphthol, 2-naphthol and 8-quinolinol)                     Herein, we purified and characterize the HypB protein from
induced local structural changes in human serum albumin                     Helicobacter pylori. The protein showed the capacity to bind


both Zn2+ and Ni2+ with a stoichiometry of one metal ion per              285, 8302-8315). Haditoxin was first identified in a cDNA
monomer. Apo-form and Zn2+-bound HypB have intrinsic                      library constructed from the venom gland tissue of O. hannah
weak GTPase activity. However, Ni2+ binding to HypB                       (FASEB J. 2007, 21, 3685-3695). The protein was isolated
significantly enhanced its GTPase activity and caused the                 from the crude venom using a two-step chromatography
dimerization of the protein. These data clearly demonstrated              and its identity was confirmed by electrospray ionization
the bifunctional roles of Ni2+ in the hydrogenase maturation              mass spectrometry and N-terminal sequencing by Edman
pathway, which served as delivery cargos as well as a regulator           degradation. Haditoxin exhibited novel pharmacology with
for HypB GTPase activity. Particually, direct Ni2+ transfer               antagonism towards muscle (αβγδ) and neuronal (α7, α3β2
from HypA to HypB protein was observed. The HypA-HypB                     and α4β2) nicotinic acetylcholine receptors (nAChRs), with
complex interfaces on HypA protein were mapped based on                   highest affinity for α7-nAChRs. The high resolution (1.5 Å)
chemical shift perturbation data and a HypA-HypB complex                  crystal structure revealed haditoxin to be a homodimer, like
model was built up using homolog modeling and docking                     κ-neurotoxins which target neuronal α3β2- and α4β2-nAChRs.
methods. Based on all the data, a GTPase regulated Ni2+                   Interestingly however, the monomeric subunits of haditoxin
delivery mechanism was proposed to elucidate the detailed                 were composed of a three-finger protein fold typical of
functions performed by the two Ni2+ chaperones.                           curaremimetic short-chain α-neurotoxins. The biochemical
Acknowledgements: We thank Research Grants Council of                     studies indicated that this protein exists as a non-covalent
Hong Kong (HKU7042/07P), RGC Collaborative Research                       dimeric species in solution and hence the protein was named
Fund (HKU1/07C and HKUST4/03C), the Area of Excellence                    Haditoxin (Hannah dimeric neurotoxin). Its structural
of UGC, and the University of Hong Kong for their support.                similarity to short-chain α-neurotoxins and κ-neurotoxins
                                                                          notwithstanding, haditoxin exhibited unique blockade of
208                                                                       α7-nAChRs (IC50 180 nM), which is recognized by neither
                                                                          short-chain α-neurotoxins nor κ-neurotoxins. This is the first
Structural and Functional Characterization of a Novel                     report of a dimeric short-chain α-neurotoxin interacting with
Homodimeric Three-finger Neurotoxin from the Venom                        neuronal α7-nAChRs as well as the first homodimeric three-
of Ophiophagus hannah (King Cobra)                                        finger toxin to interact with muscle nAChRs.
Amrita Roy1, Xingding Zhou2, D’hoedt Dieter3, Ming Zhi
Chong2, Chun Shin Foo2, Nandhakishore Rajagopalan2,                       209
Selvanayagam Nirthanan4,5, Daniel Bertrand3, J Sivaraman2,
R. Manjunatha Kini2,6                                                     Cloning, Protein Purification, and Functional
  Biological Sciences, National University of Singapore,                  Characterization of CutC, a Copper Homeostasis Protein
Block S3, #03-17, 14 Science Drive 4, Singapore, Singapore,               in Caenorhabditis elegans
117543, Singapore, 2Biological Sciences, National University              Julie Yang1, Chandra Srinivasan2, Nikolas Nikolaidis3
of Singapore, 14 Science Drive 4, Singapore, 117543,
                                                                            Chemistry and Biochemistry, California State University
Singapore, 3Dept of Neuroscience, University of Geneva, 1,                Fullerton, 800 N. State College Blvd, Fullerton, CA, 92834-
rue Michel Servet, Geneva, 1211, Switzerland, 4School of                  6866, 2Chemistry and Biochemistry, 3Biological Sciences,
Medical Science, Griffith University, Gold Coast Campus,                  California State University, Fullerton, 800 N. State College
Queensland, 4131, Australia, 5Department of Pharmacology,                 Blvd, Fullerton, CA, 92834-6866
National University of Singapore, 10 Medical Drive,                       Trace metals such as copper, zinc, and iron play important
Singapore, 117597, Singapore, 6Department of Biochemistry                 roles in protein structure and function and control proper cell
and Molecular Biophysics, Virginia Commonwealth                           function by influencing signaling pathways. The proteins
University, 1101 East Marshall Street, Richmond, VA,                      that bind and regulate the intracellular homeostasis of these
23298                                                                     metal ions are also of fundamental importance for normal cell
Snake venoms are rich source of pharmacologically active                  function. Members of the Cut superfamily have been shown to
proteins and polypeptides targeting a variety of receptors with           be involved in copper trafficking in both bacteria and humans.
high affinity and specificity. Because of their high specificity,         But whether all members of the CutC family regulate copper
some of these molecules have contributed significantly to the             homeostasis using a similar conserved mechanism remains
development of molecular probes and therapeutic agents. In                unknown. In vitro binding experiments with human CUTC
continuation of our efforts in the characterization of novel              showed a very low binding affinity suggesting that this protein
snake venom toxins (J Biol Chem. 2002, 277, 17811-17820;                  is probably an enzyme that requires copper as a cofactor. On
J Biol Chem. 2005, 280, 13137-13147; J Biol Chem. 2006,                   the other hand, in vivo studies in bacteria and the nematode
281, 29030-29041; FASEB J. 2007, 21, 3685-3695; FASEB J.                  C. elegans suggest that CutC functions to maintain copper
2009, 23, 534-545), we have identified a novel non-covalent               homeostasis and protects the cells from copper toxicity.
homodimeric neurotoxin, Haditoxin from the venom of                       Based on these results, we hypothesize that the human and
Ophiophagus hannah (King cobra) (J Biol Chem. 2010,                       the nematode CutC proteins are functionally differentiated.
                                                                          This hypothesis is indirectly supported by phylogenetic


analysis, which shows that the insect and nematode CutC                   insight into the functional domain organization and the
homologs are distantly related to their vertebrate homologs               structural mechanism of the activities of bacterial PolXs.
and their phylogenetic clustering is not in agreement with the
accepted species tree. To test our hypothesis we have cloned              211
the nematode CutC gene, produced the recombinant protein
in E. coli, and purified it using affinity chromatography. The            Characterization of the N-terminus of human copper
recombinant protein was then functionally characterized                   transporter (hCtr1)
using metal titration studies and metal binding affinity studies.         Xiubo Du, Xinghao Wang, Hongzhe Sun
Our preliminary studies suggest that the nematode protein is              the Department of Chemistry, The University of Hong Kong,
not functionally identical to its human homolog. We are also              Pokfulam Road, Hong Kong, Hong Kong (CN)
cloning CutC genes from mouse, frog, fruit fly, and several               Human high affinity copper (I) transporter 1 (hCtr1), a
bacteria and fungal species to perform comparative copper                 three transmembrane domain protein with an extracellular
binding studies. This comparative study will allow us to                  N-terminal domain, and an intracellular C-terminal domain
further elucidate the function of these proteins and explore              plays an important role in copper homeostasis in human cells.
how the function of CutC proteins evolved through time.                   In this work, we overexpressed, purified and characterized the
Support: California State University, Fullerton start up funds            N-terminal of this protein (amino acid 1-55) in vitro. dialysis
for Dr. Nikoloas Nikolaidis and California State University,              equilibrium combined with ICP-MS revealed that the protein
Fullerton funds allocated to Dr. Chandra Srinivasan.                      can bind 3 Cu+ ions per monomer. An average dissociation
                                                                          constant was determined to be KD = (K1K2K3)1/3 = 10-16 M
210                                                                       for binding of Cu+ to hCtr1_N protein via competition with
                                                                          the ligand bicinchoninic acid (BCA). The binding of Cu+ to
Structural and functional analysis of bacterial DNA                       hCtr1_N is reversible as the bound Cu+can be released in the
polymerase X involved in DNA repair processes                             presence of chelating ligand, e.g. bathocuproine disulfonate
Shuhei Nakane1, Hirohito Ishikawa1, Noriko Nakagawa1,2,                   bcs or BCA. Replacement of Met within the two Met-rich
Ryoji Masui1,2, Seiki Kuramitsu1,2                                        motifs with Ala greatly reduced the Cu+ binding affinity of
  Biological Sciences, Osaka university, 1-1 Machikaneyama-               the protein, also with a change of stoichiometry(n) value.
cho, Toyonaka, Osaka, 560-0043, Japan, 2RIKEN SPring-8                    Both gel filtration chromatography and native electrophoresis
Center, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo, 679-                     demonstrated that apo-hCtr1_N existed as a monomer in
5198, Japan                                                               solution. Binding of Cu+ to the protein induce the formation
The X-family DNA polymerases (PolXs) comprise a highly                    of a dimer, which is Met-rich motifs dependent.
conserved DNA polymerase family found in all kingdoms.                    When the histidine residues in the protein were modified by
Mammalian PolXs are known to be involved in DNA repair                    DEPC, the protein’s binding affinity for Cu+ was decreased,
and other DNA-processing pathways, especially gap-filling                 indicating hisditides may be also involved in Cu+ binding.
processes. In recent years, it has been revealed that bacteria            Considering Ag+ is similar as Cu+ in terms of charge and
have a different type of PolX. Many bacterial PolXs have a                structure, the binding property of the protein to Ag+ was
polymerase and histidinol phosphatase (PHP) domain at their               also studied. We have used isothermal titration calorimetry
C-termini in addition to a PolX core (POLXc) domain, and                  to measure the association constant (Ka) and stoichiometry
possess 3′-5′ exonuclease activity. Although both domains                 (n) values of Ag+ binding to the wild type and mutant forms
are highly conserved in bacteria, structural arrangement of               of the protein. The wild type protein binds 3 Ag+ with a
POLXc and PHP domains and the activities of each domain,                  moderate affinity (ka =105 M-1). The two Met-rich motifs
especially the roles of PHP domain in DNA repair, are                     showed similar affinity for Ag+, while both the two motifs
little known. To reveal molecular functions and structural                were mutated, no stoichiometric binding of Ag+ to the protein
information of bacterial PolX, we characterized Thermus                   was observed.
thermophilus HB8 PolX (ttPolX). The POLXc domain of                       Acknowledgement: This work was supported by
ttPolX was responsible for the polymerase and DNA-binding                 Research Grants Council of Hong Kong (HKU7512/05M,
activities, and the PHP domain was mainly responsible                     HKU7043/06P, HKU2/06C, HKU1/07C, HKU7038/08P)
for the 3′-5′ exonuclease activity. X-ray crystal structure               and the University of Hong Kong!
analysis revealed that the overall arrangement of the POLXc
and PHP domains of ttPolX was similar to that of another
bacterial PolX, however, subdomains of the POLXc domain
adopted a novel arrangement. The active-site structure of the
PHP domain was similar to that of other phosphoesterase
enzymes. This similarity suggests that the PHP domain has
a phosphoesterase activity. Our findings provide a molecular


212                                                                     Duchenne muscular dystrophy (DMD) most commonly
                                                                        through loss of protein expression. In a small sub-population
Crystal Structures of Human Thioredoxin Revealing                       of patients, missense mutations can cause DMD, Becker
Unraveled Helix and Exposed S-Nitrosation Site                          muscular dystrophy (BMD) or X-linked cardiomyopathy
Juliana The, Andrzej Weichsel, Michelle Kem, William                    (XLCM). Nearly one-half of disease-causing missense
Montfort                                                                mutations are located in actin-binding domain 1 (ABD1) of
Chemistry and Biochemistry, University of Arizona, 1041 E.              dystrophin. To test the hypothesis that ABD1 mutations cause
Lowell Street., Tucson, AZ, 85721                                       disease by impairing actin binding activity, we engineered the
S-nitrosation (also called S-nitrosylation) has been identified         K18N, L54R, D165V, A168D, L172H and Y231N mutations
as an important protein modification and implicated in                  into the full-length dystrophin cDNA and characterized the
protein regulation and possibly in the mechanism of nitric              biochemical properties of each mutant full length protein.
oxide signaling. However, not much is known about protein-              The K18N and L54R mutations result in the most severe
and structure-dependent factors for the modification due                diseases in humans and each caused a small but significant
to the labile nature of S-nitroso (SNO) groups and the lack             4-fold decrease in actin binding affinity, while the affinities
of good methods for detection. We have previously shown                 of the other mutant proteins were not significantly different
human thioredoxin-1 to be S-nitrosated at buried Cys 62                 from WT dystrophin. More interestingly, WT dystrophin was
by X-ray crystallography. Here, we describe a surprising                observed to unfold in a single-step, highly cooperative manner.
structural rearrangement in crystal structures of a C69S/C73S           In contrast, all six mutant proteins were significantly more
thioredoxin-1 double mutant. The reduced mutant structure is            prone to thermal denaturation and aggregation. Our results
similar to that of the wildtype but upon complete oxidation,            suggest that missense mutations in ABD1 may all cause loss
disulfide bonds are formed intramolecularly between the                 of function via protein instability and aggregation rather than
redox active C32 and C35 and intermolecularly between the               loss of ligand binding. However, more severe disease states
C62 residues of two monomers. This C62-C62 disulfide bond               may be due to the combinatorial effects of mutations on both
is accommodated by unraveling of helix 3 and formation                  protein aggregation and impaired actin binding.
of new interactions that result in complete exposure of the             This work was supported by the NIH Training Program in
otherwise buried residues 62 and 69. The unraveling of                  Muscle Research (AR007612) and National Institutes of
helix 3 presents insight into thioredoxin-1 dynamics and                Health grant AR042423.
suggests a mechanism for S-nitrosation of buried C62 and
the relative stability of C62-SNO conjugate. In addition,               214
we have found that organic S-nitrosothiols can form stable
N-thioorganophosphine imine products when reacted with                  Probing Potential Protein Dynamics to Elucidate
phenylphosphine compounds. We are investigating the                     the Mechanism for Enzymatic Activity in Human
reactions of S-nitrosoglutathione and thioredoxin-1 with                Immunodeficiency Virus Type-1 Protease
various phosphine compounds to examine the potential use of             Seema Mittal, Celia Schiffer
this reaction in trapping SNO-species in S-nitrosated proteins.         Biochemistry and Molecular Pharmacology, University
We hereby present an early atomic glimpse into the structural           of Massachusetts Medical School, 364 Plantation st, 970K
rearrangements that likely play a role in the mechanism of              Lazare Research Building, Worcester, MA, 01605
S-nitrosation of thioredoxin-1 and a possible alternative to            Human immunodeficiency virus Type-1 (HIV-1) protease
the biotin switch method for detecting S-nitrosated cysteines.          is a symmetric, homodimeric aspartyl protease, crucial for
This work was supported by NIH grant GM077390.                          viral maturation. Each monomer consists of 99 residues,
                                                                        40 of which are hydrophobic. From analysis of molecular
213                                                                     dynamics simulations, 19 core hydrophobic residues appear
Disease-Causing Missense Mutations in Actin Binding                     to facilitate the conformational changes that occur in HIV-1
Domain 1 of Dystrophin Induce Thermodynamic                             protease. This region has been suggested to undergo sliding
Instability and Protein Aggregation                                     motions facilitated by the exchange of hydrophobic van der
Davin Henderson, Ann Lee, James Ervasti                                 Waals contacts between the core residues. Many of these
Biochemistry, Molecular Biology and Biophysics, University              residues are away from the substrate-binding site, yet have
of Minnesota, 420 Washington Ave SE, Minneapolis, MN,                   been implicated in conferring drug resistance, the mechanism
55455                                                                   of which still remains elusive.

Dystrophin is a 427 kDa protein that interacts with actin               We hypothesize that this hydrophobic core dynamics governs
filaments at its N-terminus and the transmembrane protein               protease activity and mutations within this region that alter
dystroglycan at its C-terminus. Through these interactions              this sliding motion, will potentially change the interactions
dystrophin protects muscle from contraction-induced injury.             between hydrophobic residues and consequently impact
Mutations in the dystrophin gene cause the lethal disease               the catalytic activity of the protease. To determine whether


locking the hydrophobic core using covalent chemistry                      Structural studies will exhibit helpful details that can explain
compromises protease activity, we have engineered three                    the functional evidence demonstrating interactions between
protease variants with novel disulfide bridges within the                  Pbx1 and Pdx1, including defining the protein-protein
hydrophobic core region. Each variant was selected to test                 interaction interfaces, the relative orientation of the proteins
the contribution of different region of the hydrophobic core               in the complex, and the protein-DNA interactions. The
in the hydrophobic sliding.                                                structural model of the Pbx1-Pdx1-DNA complex will be
We have determined 3 crystal structures of these protease                  compared with the structure of Pdx1-DNA alone. The Pdx1-
mutants under reducing conditions, where the incorporated                  DNA structure showed that the homeodomain has flexibility
cysteines are in the reduced form. We have also determined                 in binding DNA. We anticipate that the interaction with Pbx1
one structure in oxidizing conditions, thus successfully                   will select a unique conformation of Pdx1, and unique DNA
locking down the core with disulfide bonds. The kinetic                    contacts, in the Pbx1-Pdx1-DNA complex.
studies on these variants in oxidized and reduced conditions
and detailed structural analysis of these structures is currently
underway to help elucidate the effects of loss of core flexibility
                                                                           Protein-Based Biomaterials
on protease function.                                                      (216 – 219)
This research was supported by the NIH grants (P01-
GM66524 and R01GM064347).                                                  216
                                                                           A protein-based artificial retina: Optimization of
215                                                                        chemically oriented Bacteriorhodopsin films for electrical
Crystallization of the Pbx1-Pdx1-DNA complex on a                          stimulation of retinal cells
somatostatin-specific promoter sequence                                    Matthew Ranaghan1, Nicole Wagner1, Megan Sandberg2,
Dongli Wang, Robert Rose                                                   Daniel Sandberg2, Rickinder Grewal1, Rekha Rangarajan1,
Biochemistry Department, North Carolina State University,                  Anu Nellisery1, Ralph Jensen3, Robert Birge1,2
128 Polk Hall, Raleigh, NC, 27695
                                                                            Department of Molecular and Cell Biology, University of
                                                                           Connecticut, 91 North Eagleville Road, Unit 3125, Storrs, CT,
The homeodomain transcription factor Pdx1 fulfills multiple
                                                                           06269, 2Department of Chemistry, University of Connecticut,
functions in the development of the pancreas. In the mature
                                                                           55 North Eagleville Road, Storrs, CT, 06269, 3Center for
pancreas, Pdx1 regulates the expression of two endocrine
                                                                           Innovative Visual Rehabilitation, VA Boston Healthcare
hormones: insulin in beta cells and somatostatin in delta cells.
                                                                           System, 150 South Huntington Avenue, Boston, MA, 02130
In order to satisfy these different roles, Pdx1 cooperates with
different transcription factors. Pdx1 and E47/NeuroD regulate              Sight is critical to the human experience and the enjoyment
the insulin enhancer in beta cells. Pdx1 interacts with Pbx1 in            of life. Millions of people, however, are afflicted with visual
delta cells to regulate the somatostatin promoter.                         disorders for which few treatments are available. Much of
                                                                           the current effort in vision restoration targets photoreceptor
In order to understand the mechanism by which Pdx1 targets
                                                                           diseases, such as macular degeneration and retinitis
different genes during development and in the mature
                                                                           pigmentosa, that leave the neural network of the retina
pancreas, we are studying the interaction of Pdx1 and Pbx1
                                                                           intact. Although promising artificial retina technologies are
on the somatostatin promoter.
                                                                           under development, most current designs are either highly
Pdx1 regulates somatostatin expression in delta cells via 2                invasive or require external power sources to drive the
somatostatin-specific elements, termed TSEI and TSEII.                     device. Conversely, our design explores the potential of a
The TSEII element of the somatostatin promoter recognizes                  self-contained, protein-based artificial retina. Specifically,
a heteromeric complex composed of Pdx1 and Pbx1. Pbx1,                     Bacteriorhodopsin (BR) is a photoactive membrane protein
which is essential for normal pancreatic development and                   that exhibits analogous photophysical properties to that of
function, is also a member of homeodomain transcription                    the native photoreceptors in the eye. This protein is very
factor family. Specificity of Pdx1 action in delta cells may               well characterized, often the paradigm of a vast array of
in part be dictated by the ability of potential target promoter            biophotonic devices, and thus well suited for this application.
sites to recognize the Pbx1-Pdx1 heteromeric complex.                      Such a device, although currently limited to photoreceptor
Our immediate goal is to co-crystallize the Pbx1-Pdx1-DNA                  diseases, is necessary because of the increasing average human
complex and to determine the structure. We have prepared                   lifetime and number of those faced with related ailments.
the complex with different DNA fragments originated from                   Our design is composed of an electrostatically constructed,
the wild-type TSEII element and the TSEII element with a                   highly-ordered and uniformly oriented protein film that will
point mutation. We have obtained small crystals, which are                 electrically stimulate the ganglion or bipolar cells when
currently being optimized.                                                 activated via a light stimulus. Films comprised of 200 layers
                                                                           of native BR are capable of producing 800 mV. This value is


roughly half the required voltage for stimulation of a rabbit         218
ganglion cell, as determined in preliminary experiments with
electrically simulated BR pulses. Mutants, which produce up           Drug Delivery System for Poorly Water-soluble Drugs by
to a 6-fold increase from the wild-type photovoltaic signal,          Lipocalin-type Prostaglandin D Synthase
are now being employed in analogous films for testing.                Ayano Fukuhara1,2, Hidemitsu Nakajima3, Katsuaki
                                                                      Inoue4, Yuya Miyamoto1,2, Shigeru Shimamoto2,5, Shigenori
217                                                                   Nishimura1, Tadayasu Ohkubo5, Tadayoshi Takeuchi3,
                                                                      Takashi Inui1
Preparation of Liposomal Nanovesicles with Influenza                  1
                                                                       Graduate School of Life and Environmental Sciences,
Hemagglutinin Activity                                                Osaka Prefecture University, 1-1, Gakuen-Cho, Nakaku,
Min-Ying Wang1, Gary Ro-Lin Chang1, Su-yuan lai3, Pei-                Sakai, Osaka, 599-8531, Japan, 2Resarch Fellow of the Japan
Tzu Chu2, Hsiao-Wei Wen2                                              Society for the Promotion of Science, 8 Ichibancho, Chiyoda-
  Graduate Institute of Biotechnology, 2Department of Food            ku, Tokyo, 102-8472, Japan, 3Graduate School of Life and
Science and Biotechnology, National Chung Hsing University,           Environmental Sciences, Osaka Prefecture University, 1-58,
250, Kuo Kuang Rd., Taichung, 402, Taiwan, 3Department                Ouraikita, Rinku, Izumisano, Osaka, 598-8531, Japan, 4Japan
of Food Science, Central Taiwan University of Science and             Synchrotron Radiation Research Institute, 1-1-1, Kouto,
Technology, No.666, Buzih Road, Beitun District, Taichung             Sayo-cho, Sayo-gun, Hyogo, 679-5198, Japan, 5Graduate
City, 40601, Taiwan                                                   School of Pharmaceutical Sciences, Osaka University, 1-6,
Influenza is a highly contagious disease which causes                 Yamadaoka, Suita-shi, Osaka, 565-0871, Japan
numerous morbidities and mortalities worldwide each                   [Introduction] Lipocalin-type prostaglandin D synthase
year. The outbreak of swine H1N1 influenza recently has               (L-PGDS) is abundantly expressed in the central nervous
raised the public panic of imminent pandemic. Prophylactic            system of various mammals. L-PGDS is a unique dual
vaccination is the most effective way to reduce influenza             functional protein acting as both a PGD2-synthesizing
virus infection. In this study, a recombinant baculovirus             enzyme and a secretory lipid-transporter protein. L-PGDS
expressing the ectodomain of influenza hemagglutinin                  can bind a wide variety of hydrophobic small ligands. So far,
(HA) was constructed to express as a soluble and secreted             hydrophobic drugs have been frequently dropped out from
protein. The secreted protein (eHA7h) was purified with a             the development in the preclinical stage because the insoluble
purity about 97% by ammonium sulfate precipitation and                characteristics fail to validate their potencies in vivo. Here
immobilized metal ion affinity chromatography (IMAC).                 we propose a novel drug delivery system (DDS) using
The purified eHA7h protein was functionalized and                     L-PGDS for hydrophobic and poorly water-soluble drugs
conjugated to thiolated-liposomal nanovesicles, which were            such as diazepam (DZP), a major benzodiazepine anxiolytic
composed of dipalmitoylphosphatidylethanolamine (DPPE),               drug, and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione
phosphatidylcholine (PC) and cholesterol. The eHA7h-tagged            (NBQX), an AMPA receptor antagonist.
liposomes exhibited a strong hemagglutination response and            [Results] We measured the fluorescence quenching of the
the capacity to bind the receptors on MDCK cells, which               intrinsic tryptophan residues of L-PGDS to investigate the
indicated that the recombinant eHA7h was properly folded and          binding affinity of DZP and NBQX. As a result, L-PGDS
biologically active, suggesting that displaying recombinant           showed the ability to bind both DZP and NBQX with
HA ectodomain on the surface of synthetic liposomes can be            the dissociation constants of 84 and 12 μM, respectively.
applied develop an influenza vaccine.                                 To reveal further details of the binding mechanism, we
                                                                      investigated the structures of DZP/L-PGDS complex and
                                                                      NBQX/L-PGDS complex by small-angle X-ray scattering
                                                                      (SAXS) and multi-dimensional NMR measurements.
                                                                      SAXS measurements revealed that L-PGDS has a globular
                                                                      shape and becomes compact by 1.2 Å and 2.0 Å in radius
                                                                      of gyration on binding DZP and NBQX, respectively. NMR
                                                                      experiments revealed that L-PGDS holds these drugs within
                                                                      the hydrophobic cavity of L-PGDS. By the addition of 500
                                                                      μM L-PGDS, the solubility of both DZP and NBQX in PBS
                                                                      were increased approximately 7- or 2-fold, respectively. To
                                                                      validate the availability of L-PGDS as a drug delivery vehicle
                                                                      in vivo, we performed two paradigms: the oral administration
                                                                      of DZP/L-PGDS complex in pentobarbital-induced mice
                                                                      and the intravenous treatment of NBQX/L-PGDS complex
                                                                      in ischemic gerbils. Eventually, DZP/L-PGDS complex


augmented the duration of pentobarbital-induced loss of                 Protein-Protein and Protein-
righting reflex, and NBQX/L-PGDS complex showed
the protective effect on delayed neuronal cell death at the             Ligand Interactions (220 – 245)
hippocampal CA1 region, suggesting that these complexes
showed the prospective effect of each drug.                             220
[Conclusion] Taken together, these results demonstrate that             Comparing two methods for determining the binding
L-PGDS is a beneficial delivery vehicle for poorly water-               affinity of an antibody to a cell surface expressed target
soluble drugs. The novel DDS could enable to facilitate of the          Susanne Scesney, Leena Kalghatgi, Eve Barlow, Mary
pharmaceutical and clinical developments of various water-              Leddy, Suju Zhong, Xiaoqing Lu, Denise Karaoglu
insoluble drugs.                                                        Abbott Bioresearch Center, 100 Research Drive, Worcester,
                                                                        MA, 01605
                                                                        The binding affinity or Kd of an antibody to its target is a
Biological Protein Crystals: A Novel Platform for Cellular              critical characteristic whether the antibody is used as an
Imaging and Protein Delivery                                            assay reagent or as a therapeutic drug. For soluble targets,
Manoj Nair1, Marianne Lee1, Julie Wallace2, Richard Burry3,             where recombinant proteins can be utilized, many methods
Michael Ostrowski2, Michael Chan1                                       such as ELISA and BIAcore are available for determining the
  Biochemistry, The Ohio State University, 484 West 12th                Kd. While these methods are sufficient for soluble targets, a
Ave, Rm 812, Columbus, OH, 43210, 2SBS-Molecular                        cell-based binding affinity method is required for a plasma
and Cellular Biochemistry, The Ohio State University, 460               membrane expressed target: binding a to soluble recombinant
West 12th avenue, Rm 810, Columbus, OH, 43210, 3SBS-                    extracellular domain construct does not take into account
Neuroscience, The Ohio State University, 460 West 12th                  receptor number or other accessory molecules present on the
Avenue, Rm 277, Columbus, OH, 43210                                     cell which can also affect the Kd. We compared two methods
While it is generally accepted that the major role of protein           for measuring the Kd of an antibody to a cell surface molecule:
crystals in biology is for the structure determination of               a traditional receptor binding assay, using Europium or 125I
proteins, protein crystals also have potential applications as          labeled antibody, and an electrochemiluminescence method
biomaterials. For example, they can serve as a framework                where the cells are coated to a carbon surface. Results obtained
to stabilize proteins, or as a carrier for delivery of enzyme           from these methods will be compared. The advantages and
therapeutics. While studies of protein crystals as biomaterials         disadvantages of both methods will be discussed.
have mostly focused on crystals obtained by in vitro
crystallization, these approaches have shortcomings in the              221
general uniformity of the crystals produced, and require                Specificity and Cooperativity at β-lactamase Position 104
extensive screening of suitable crystallization conditions for          in TEM-1/BLIP and SHV-1/BLIP interactions
each new protein targeted. Moreover, most protein crystals              Melinda Hanes1, Kimberly Reynolds2, Case McNamara3,
dissolve when removed from the precipitating agent and                  Partho Ghosh3, Robert Bonomo4, Jack Kirsch5, Tracy
thus require the use of exogenous crosslinking agents that              Handel1
can affect protein function and toxicity. A novel solution for          1
                                                                         Skaggs School of Pharmacy, University of California San
producing protein crystals that circumvents many of these               Diego, 9500 Gilman Drive, La Jolla, CA, 92093, 2Department
limitations is provided by the gram positive soil bacterium             of Pharmacology, University of Texas Southwestern
Bacillus thuringiensis. This unusual bacterium produces                 Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390,
crystal proteins or Cry proteins that assemble in vivo to form          3
                                                                         Chemistry and Biochemistry, University of California San
protein crystals of fairly uniform size (0.5-1.0 µm). Notably,          Diego, 9500 Gilman Dr., La Jolla, CA, 92093, 4Research
our structural studies of Cry proteins fused to the fluorescent         Service, Louis Stokes Veterans Affairs Medical Center,
protein GFP (or its variants e.g. CFP, YFP, mCherry),                   10701 East Blvd., Cleveland, OH, 44106, 5Biochemistry and
led to the exciting observation that B. thuringiensis cells             Molecular Biology, University of California Berkeley, 176
overexpressing Cry-GFP fusion proteins form fluorescent                 Stanley Hall # 3220, Berkeley, CA, 94720
crystals demonstrating that the GFP subdomain still folds
property and that it can fit into the Cry crystal framework.            Describing how cooperative interactions contribute to
Here we describe the facile production and purification of              affinity and specificity is challenging, and moreover, these
these Cry-GFP crystals, and illustrate their application as an          effects are complicated to experimentally characterize.
imaging agent for cellular studies both in vitro and in vivo.           β-Lactamase/BLIP complexes have been useful platforms
Our progress on the encapsulation of other proteins and their           for understanding mechanisms for affinity and specificity
applications will also be described.                                    of protein-protein interactions. BLIP recognizes the TEM-
                                                                        1 and SHV-1 β-lactamases with Ki values of 3 nM and 2
                                                                        µM, respectively. Previous study has identified β-lactamase


position 104 as a specificity determinant in β-lactamase/              negatively regulates all iGluRs. Our recent findings show
BLIP interactions: TEM-1 E104 participates in a salt bridge            that both neurosteroids bind to the amino terminal domain
with BLIP K74, whereas the corresponding SHV-1 D104                    (ATD) and ligand binding S1S2 domain of the NMDA NR2B
does not. Recent work demonstrated that the BLIP mutation              subunit but only bind to the ATD of the NR2D subunit. We
E73M facilitates a similar salt bridge between SHV-1 D104              hypothesize that PS binding site in the ATD is inhibitory,
and BLIP K74. Here, the cooperativity between the Glu/Lys              while that in the ligand binding S1S2 domain is potentiating.
or Asp/Lys pair and a neighboring residue was investigated             Our lab has also discovered a neurosteroid binding domain in
using double mutant cycle analysis for TEM-1/BLIP E73M                 the S1S2 domain of the GluR2 subunit of the AMPA receptor.
and SHV-1/BLIP E73M interfaces, respectively. In the                   Intrinsic and extrinsic fluorescence studies will be presented
absence of the proximal sidechain F142 in BLIP (F142A), the            that confirm the binding of PS and PREGAS to the inhibitory
Asp/Lys salt bridge contributes unfavorably to SHV-1/BLIP              ATD of the NMDA NR2D subunit and the lack of binding
affinity by1.3 kcal/mol, and the Glu/Lys pair is energetically         to the S1S2 domain. In addition, binding studies to both
neutral in the TEM-1/BLIP interface. However, BLIP                     the S1S2 and ATD of the GluR2 subunit will be presented.
F142 cooperatively stabilizes both interactions and the salt           Data from KD studies, Stern-Volmer analysis, and circular
bridging partners contribute 1.6 kcal/mol and 0.9 kcal/mol, in         dichroism spectroscopy will be presented to help characterize
TEM-1/BLIP and SHV-1/BLIP, respectively. Furthermore,                  the binding.
we structurally characterized the SHV-1 D104E/BLIP                     Funded by National Science Foundation
complex (∆∆G value of -4.4 kcal/mol) and the resulting 1.6
Å resolution crystallographic structure confirms a TEM-like            223
salt bridge between D104E and BLIP K74. Additionally, the
analogous mutation in TEM-1, TEM-1 E104D displays SHV-                 Understanding the potentiation of NMDA iGluRs by
like affinity for BLIP. Hence, β-lactamase position 104 is             pregnenolone sulfate
the sole specificity determinant between the two interfaces.           Krasi Cameron, Sarah Rhoads, Lisa Gentile
Here, we describe how a single mutation mediates large                 Chemistry, University of Richmond, 28 Westhampton Way,
perturbations in interface stability through involvement in a          Richmond, VA, 23173
cooperative interaction network. This work demonstrates the            AMPA, NMDA, and kainate receptors belong to the ionotropic
importance of hot spots in determining specificity, and the            glutamate (iGluR) family of receptors. Upon binding to the
role of cooperativity in such interactions.                            ubiquitous fast excitatory neurotransmitter glutamate, these
                                                                       post synaptic neural membrane channels activate, therefore
222                                                                    they play an important role in synaptic plasticity, memory
                                                                       and learning. Our research focuses on understanding how
Understanding the inhibitory regulation of AMPA and
                                                                       these receptors are being regulated for potential applications
NMDA iGluRs by sulfated neurosteroids
                                                                       in conditions such as Alzheimer’s, Huntington’s and
Krasi Cameron, David Fanelli, Beth Pollard, Emily Bartle,
                                                                       Parkinson’s disease.
Lisa Gentile
Chemistry, University of Richmond, 28 Westhampton Way,                 The NMDA receptors are most often heterotetramers
Richmond, VA, 23173                                                    composed primarily of two glycine binding NR1 subunits
                                                                       and two glutamate binding NR2 subunits. The NR1 subunit
AMPA, NMDA, and kainate receptors belong to the
                                                                       is encoded by eight alternatively spliced versions of the same
ionotropic glutamate (iGluR) family of receptors located
                                                                       gene, while the NR2 subunit is encoded by four separate genes
in the post-synaptic neural membrane. Upon binding to
                                                                       (NR2A-D). The data presented here is aimed at understanding
glutamate, a major fast excitatory neurotransmitter, these
                                                                       the potentiation of NMDA receptors containing NR2A or
channels activate, consequently they play an important role
                                                                       NR2B subunits by the endogenous neurosteroid pregnenolone
in synaptic plasticity, memory and learning. Mis-regulation
                                                                       sulfate (PS). This neurosteroid has inhibitory action on
of these receptors has been linked to many neurodegenerative
                                                                       NMDA receptors containing NR2C or NR2D subunits and all
diseases, therefore our research focuses on understanding the
                                                                       other iGluRs. Intrinsic and extrinsic fluorescence studies will
receptors’ binding regulation for potential applications in
                                                                       be presented that confirm the binding of the NMDA NR2B
conditions such as Alzheimer’s and Parkinson’s disease.
                                                                       S1S2 and amino terminal domain (ATD) to PS. Unlike the
The data presented here is aimed at understanding the                  NMDA NR2B subunit, the NMDA NR2D subunit binds to PS
inhibitory regulation of AMPA and NMDA receptors by                    only in the ATD allowing us to hypothesize that PS binding
the endogenous neurosteroids pregnenolone sulfate (PS)                 to the ATD is inhibitory, while binding to the S1S2 domain
and 3á-hydroxy-5â-pregnan-20-one sulfate (PREGAS). PS                  is potentiating. Data from circular dichroism spectroscopy,
potentiates NMDA receptors containing NR2A or NR2B                     isothermal titration calorimetry, and Stern-Volmer analysis
subunits, while inhibiting NMDA receptors containing NR2C              will be presented to help characterize the binding site of
or NR2D subunits and all other iGluRs. Likewise, PREGAS                the neurosteroid in each of the NMDA NR2B domains and


explain the potentiating action of PS on NMDA receptors                   225
containing NR2B subunits.
                                                                          Genome-Wide Structural Modeling of Protein-Protein
Funded by National Science Foundation                                     Interactions
                                                                          Petras Kundrotas, Zhengwei Zhu, Ilya Vakser
224                                                                       Bioinformatics, University of Kansas, 2030 Becker Dr.,
Mutational and Structural Analysis of the P2’ Specificity                 Lawrence, KS, 66045
of Mesotrypsin Toward Kunitz Protease Inhibitors                          Rapid development of modeling approaches and advances
Mohd Salameh1, Alexei Soares2, Evette Radisky1                            in experimental structure determination enable structural
  Mayo Clinic Cancer Center, Mayo Clinic, 4500 San Pablo                  modeling of protein-protein interactions (PPI) for entire
rd, Griffin cancer Research bldg., Jacksonville, FL, 32224,               genomes. A hierarchy of modeling approaches for high-
  Biology Department, Brookhaven National Laoratory,                      throughput structural modeling of PPI is being implemented.
National Synchrotron Light source, Upton, NY, 11973                       At the current stage of development (template-based
Kunitz protease inhibitors, including bovine pancreatic                   algorithms only) it was applied to all interactions in DIP and
trypsin inhibitor (BPTI) and the Kunitz domain of the amyloid             BIND databases (127,124 non-redundant PPI), covering 771
precursor protein (APPI), inhibit trypsin and other serine                genomes from the entire universe of life. For 14,635 PPI, the
proteases by binding to the enzyme very tightly in a substrate-           template complexes were found and 3D models were built
like manner, but resisting proteolysis. Mesotrypsin is an                 by sequence homology (homology docking). For additional
unusual human trypsin that is uniquely resistant to inhibition            26,449 interactions, the monomer models were independently
by Kunitz protease inhibitors, cleaving and inactivating these            built by sequence homology and the search for the template
inhibitors at accelerated rates. We have found that different             complexes was performed by structure alignment. The
Kunitz protease inhibitors differ substantially in affinity and           models quality was assessed by comparison of the structural
susceptibility to mesotrypsin: BPTI is bound very weakly                  characteristics of the modeled interfaces with those of
and cleaved slowly, while APPI is a highly specific substrate,            the crystallographically determined complexes. Template
bound tightly and cleaved rapidly. Here, we define the P2′                complexes from organisms different from the target complex
substrate specificity of mesotrypsin through kinetic analysis             organism substantially increased the number of structural
and X-ray crystallography using mutants of APPI and BPTI.                 models (e.g., PPI from human modeled using the template
We find that introduction of acidic residues Asp or Glu at                PPI from Archaea). The part of PPI accounted for by the
the P2′ position increases specificity kcat/Km by increasing              template-based methods (structural coverage) is substantially
the catalytic turnover kcat. Introduction of Gly at the P2′               lower for organisms overrepresented in the existing PPI
position also increases specificity kcat/Km, but in this case the         databases. For example, out of 38,101 interactions known in
improvement is implemented through tighter binding affinity,              fungi, 37,641 are from the baker’s yeast, with the structural
coinciding with an alternative conformation of mesotrypsin                coverage ~35%, whereas for the remaining 46 fungi
active site residue Arg-193 in the crystal structure of the               organisms the coverage is ~75%. The results are implemented
complex. By mutating the binding loop of BPTI at two                      in GWIDD database (, a user-
positions, incorporating Arg at the P1 position and Gly at the            friendly comprehensive resource for PPI structural modeling
P2′ position, we have generated an inhibitor, BPTI-K15R/                  on genome scale.
R17G, with 1,400-fold improved mesotrypsin affinity relative
to BPTIwt; this inhibitor combines strong mesotrypsin                     226
affinity (Ki=10 nM) with proteolytic stability (kcat=0.000051             Modeling of protein-protein interactions by structural
s-1). Our studies with APPI and BPTI show that Kunitz                     similarity at the interfaces
inhibitor resistance to proteolysis and binding affinity are              Petras Kundrotas, Rohita Sinha, Ilya Vakser
orthogonal properties that can be independently manipulated.              Bioinformatics, University of Kansas, 2030 Becker Dr.,
Furthermore, as mesotrypsin has been implicated in cancer                 Lawrence, KS, 66045
progression, our studies provide support for the feasibility of
engineering Kunitz inhibitors as tools to define the specific             Protein-protein interactions (PPI) play the key role in all
role of mesotrypsin in cancer models, and potentially as                  aspects of cell functioning. The structural aspects of PPI
novel mesotrypsin-targeted therapeutics.                                  are essential for the description of life processes at the
                                                                          molecular level. Rapid accumulation of the experimental
                                                                          data on protein-protein complexes drives the paradigm shift
                                                                          in protein docking from template free to template based
                                                                          approaches. Homology docking, based on the sequence
                                                                          similarity between the target and the template, can account
                                                                          for ~ 20% of known PPI. If the templates for the complex
                                                                          are not available, but the structure of the target monomers is


known, docking by local structural alignment may provide              Preclinical models of prostate cancer show that hepsin
an adequate solution. Such algorithm was developed based              promotes tumor progression and that inhibition of its
on the structural comparison of monomers to known co-                 enzymatic activity inhibits invasive growth and metastasis.
crystallized interfaces. A library of templates was compiled,         In an effort to develop specific antibody inhibitors against
consisting of 11,932 interface fragments, extracted from the          hepsin, we performed phage display with an antibody Fab
asymmetric and biological units in PDB. The procedure was             library of restricted chemical diversity at the complementary
validated on the DOCKGROUND docking benchmark sets:                   determining regions (CDR). Among several binders the lead
99 unbound complexes and the extended set of 372 bound                antibody (Fab25) showed the highest binding affinity for
complexes. The results were optimal with the interfaces               hepsin (KD 7.6 nM) as determined by biolayer interferometry
defined by 12 A distance cutoff. Higher-accuracy models               using an OctetRED-384™ instrument. Fab25 has an
(i-RMSD < 5 A) were found in top 10 predictions for 25%               unusually long CDR-H3 loop (21 amino acids) containing
(unbound set) and 33% (extended bound set) of targets. In             one Arg and three Lys residues, which may constitute
19% of cases (extended set) sequence identities between               potential hepsin cleavage sites. However, despite prolonged
target and template complexes were < 20%, making those                incubation no proteolytically processing of Fab25 by hepsin
templates hardly detectable by sequence-based homology                was observed. Fab25 is highly specific for hepsin as indicated
methods. Compared with the full structure alignment, the              by the complete lack of inhibitory activity towards a panel
partial structure alignment succeeded in a significant number         of related trypsin-like serine proteases. In enzyme assays
of targets on which the full-structure alignment failed and           with a synthetic substrate (S2765), Fab25 showed complete
yielded higher-accuracy models with systematically lower              inhibition of hepsin activity (Kiapp 4.1 nM) and Eadie-
i-RMSD values. The results indicate that the structure                Hofstee plots inferred that the inhibition mechanism was
alignment techniques provide a much needed addition to the            non-competitive. In competition binding experiments using
docking arsenal, with the combined structural alignment and           biolayer interferometry, Fab25 competed with two active
template free docking success rate significantly surpassing           site-directed inhibitors, the HAI-1 derived Kunitz domain
that of the template free docking alone.                              inhibitor KD1 and the peptidic hepsin inhibitor Ac-Lys-
                                                                      Gln-Leu-Arg-chloromethyl ketone. This suggested that the
227                                                                   Fab25 binding site includes structural elements at or around
                                                                      the hepsin active site region. Fab25 completely blocked
Thermodynamic basis of nucleotide discrimination in                   hepsin enzymatic activity towards known macromolecular
the PBCV-1 mRNA capping enzyme: a thermodynamic                       substrates. In addition, Fab25 also inhibited full-length cell
integration study                                                     surface expressed hepsin in an assays system that measured
Robert Swift1, Rommie Amaro1,2                                        pro-HGF processing by hepsin-overexpressing LnCaP cells.
  Pharmaceutical Sciences, University of California, Irvine,          These findings demonstrate that the phage-derived Fab25 is
University of California at Irvine, Irvine, CA, 92697,                a highly specific and potent inhibitor of hepsin enzymatic
  Computer Science, University of California, Irvine,                 activity and, thus, may constitute a valuable tool to further
University of California, Irvine, Irvine, CA, 92697                   investigate the therapeutic potential of targeting hepsin in
The PBCV-1 mRNA capping enzyme is a GTP-specific                      prostate cancer.
enzyme closely related to the PBCV-1 DNA ligase, an ATP-
specific enzyme. By using thermodynamic integration                   229
and molecular dynamic simulations, the subtle binding
site differences between the two enzymes are analyzed in              Structural and thermodynamic determinants of
context of nucleotide specificity. We show that nucleotide            noncovalently oligomeric protein ligands
specificity arises from a small set of non-conserved binding-         Gregory Poon, Alaina Forbes
site residues.                                                        Pharmaceutical Sciences, Washington State University, P.O.
                                                                      Box 646534, Pullman, WA, 99164
228                                                                   Incorporation of compact oligomerization domains is an
                                                                      efficient method of generating oligomeric protein ligands.
Identification of the antibody phage display-derived                  This strategy offers the advantages of thermodynamic
Fab25 as a potent and specific inhibitor of serine protease           self-assembly and potential multivalency at a low cost for
hepsin                                                                size. It also avoids the specialized chemistry required for
Rajkumar Ganesan, Yingnan Zhang, Paul Moran, Sachdev                  introducing covalent crosslinks and is equally applicable for
Sidhu, Daniel Kirchhofer                                              both synthetic peptides and recombinant proteins. Several
Protein Engineering, Genentech, 1 DNA Way, MS 27, South               examples in the literature have demonstrated the increased
San Francisco, CA, 94080                                              affinity and biological activity of these oligomeric proteins
Hepsin, a type II transmembrane serine protease, is one               compared to their monomeric counterparts. Although the
of the most highly upregulated genes in prostate cancer.              structural and thermodynamic determinants that confer


such enhancements have remained largely unexplored,                    231
we hypothesize that they offer significant potential for
optimization. To this end, we are developing a cell-based              Hit Validation by Direct Measurement of Protein
model aimed at elucidating the quantitative contributions              Conformational Change
of these factors, using a HER2-directed affibody (ZHER2:342)           Mark Gostock1,2, Jon Popplewell3, Gerry Ronan3, Marcus
as model ligand. More specifically, we have genetically                Swann3
fused ZHER2:342 to several series of oligomerization domains
                                                                        Farfield Group, 4514 Plummer Street, Pittsburgh, PA,
derived from the tetramerization domain of human p53                   15201, 2Farfield Group, Electraway, Southmere Ct, Crewe,
and coiled coil domain of GCN4. Each series differs from               CW1 6GU, United Kingdom, 3Fafield Group, Electraway,
each other in terms of molecularity and topology, while                Southmere Ct, Crewe, CWI 6GU, United Kingdom
members within each series are structurally homologous but             Label free technologies are now commonplace within the drug
thermodynamically variable. We are characterizing the cell-            discovery process with candidate selection either by affinity
binding and internalization of each (radiolabeled) oligomeric          or kinetic response or a combination of both. High affinity
ligand into live, HER2-positive SK-OV-3 cells at 4°C and               however is no assurance of specificity towards the target’s
37°C respectively, using HER2-negative MDA-MB-231                      active site, and downstream biophysical characterization of
cells as control. By correlating the cell-based results with           the hit (hit validation) is invariably required before further
structural and thermodynamic parameters (topology, linker              optimization steps are justified.
length, oligomeric stability), we are constructing a design            In this work we have used Dual Polarisation Interferometry,
profile for generating highly active oligomeric ZHER2:342-             a label free interactions instrument, to directly measure the
based ligands. This approach should also be applicable to              conformational change associated with small molecules
developing noncovalently oligomeric derivatives of other               binding to a kinase target. Comparison to the conformational
targeted ligands of biochemical interest.                              response to an ATP analog (AMP) demonstrate that the
                                                                       response can clearly differentiate between binding at the ATP
230                                                                    site and allosteric distortion of the target.
Improvement of hit ratio using combination of in silico                As structure and structural change is measured from which
and SPR screening for anti-prion compounds                             kinetic data and equilibrium constants can be obtained,
Hironori Nakamura, Yuji Kamatari, Junji Hosokawa-Muto,                 conformation may be trivially linked to function in a single
Kazuo Kuwata                                                           set of high-content measurements. This allows the analyst
Center for Emerging Infectious Diseases, 1-1, Yanagido,                to understand the mechanism of action of binders, and
Gifu, 501-1194, Japan                                                  differentiate between specific and non-specific interactions,
Prion diseases are fatal neurodegenerative diseases such as            and candidates with differential binding modes. It is
Creutzfeldt-Jakob disease in humans and bovine spongiform              postulated that such conformational characterization of
encephalopathy in cows. There is no established treatment for          interactions could greatly narrow the ranks of candidates for
human prion diseases at this stage. Here we targeted compounds         lead optimization.
which bind to the cellular form of prion protein (PrPC) and
stabilize it to prevent the misfolding of prion protein.               232
In our previous study, 24 anti-prion compounds were found              The production of protein-protein complexes in high-
among 204 compounds selected by in silico screening [1].               throughput manner for functional and structural
Those hit compounds were identified by bioassay using the              characterization
Fukuoka-1 strain-infected neuronal mouse cell line.                    Gyorgy Babnigg, Robert Jedrzejczak, Boguslav Nocek,
In this study, we examined the binding affinity with PrPC              Adam Stein, William Eschenfeldt, Norman Marshall, Alicia
for all the assayed 204 compounds with surface plasmon                 Weger, Ruiying Wu, Lucy Stols, Kimberly Buck, Andrzej
resonance (SPR) using BIAcore T100 at a compound                       Joachimiak
concentration of 10μM. Although some compounds showed                  MCSG, Biosciences Division, Argonne National Laboratory,
relatively-large response without anti-prion activity, 14 hit          9700 S. Cass Ave, Bldg 202, Argonne, IL, 60439
compounds were found among the top 50s in the ranking list             Protein-protein complexes play a key role in the majority
of maximum amplitude of BIAcore response. Hit ratio for                of cellular functions. A sizeable fraction of the interactome
the 50 compounds was 28.0% although the original hit ratio             is stable and can be isolated for functional studies and
for 204 compounds was 11.8%. This indicates that the further           structural characterization via X-ray crystallography.
selection by BIAcore response after the primary selection by           3-dimensional structures provide a critical insight into the
docking simulation improves the hit ratio for bioassay.                functions and interactions of these molecular machines at the
1 Hosokawa-Muto et al.         (2009)    Antimicrob Agents             atomic level. The biochemical characterization of enzyme
  Chemother 53: 765-771                                                complexes purified form the native host might be hampered


by contaminating proteins. Structural characterizations of             toward the development of chemical probes. These probes
protein-protein complexes directly isolated from the native            are developed to inhibit a protein selectively in a cell and can
host are limited only to highly abundant complexes. These              be a powerful tool to probe the protein target, investigate its
problems can be circumvented via the recombinant expression            biological role and assess its suitability as a pharmaceutical
of the interacting partners. Several approaches have been              target
described for the production of large quantities of protein
complexes, such as purification and mixing the individual              234
components, co-expressing the interactive partners in a
bicistronic vector, or by the use of recombination systems. At         Energetics-based target identification by prefractionation
present most of these techniques are either not economical or          and pulse proteolysis
compatible with high-throughput (HTP) operations.                      Chiwook Park, Youngil Chang
                                                                       Department of Medicinal Chemistry and Molecular
We have recently developed a technique amenable to HTP                 Pharmacology, Purdue University, 575 Stadium Mall Dr,
operations for expression of a large number of protein-protein         West Lafayette, IN, 47907
complexes using a standard Ligation Independent Protocol
(LIC). We employed two HTP cloning strategies for a selected           Identification of small molecule targets is a critical but still
set of 384 protein-protein complexes: the co-expression                challenging step in chemical genomics and drug discovery.
of the interacting partners as an operon or as a bicistronic           Energetics-based target identification is an approach to identify
cassette. We have processed more than 50 complexes and                 proteins interacting with small molecules by exploiting the
obtained crystals for more than 20 of them. Structures of              change in conformational energetics of the target proteins
3-oxoadipate coA-transferase and molybdopterin converting              upon ligand binding. Different from the traditional affinity-
factor from Helicobacter pylori have been determined. We               based separation methodology, energetics-based target
demonstrate that our target selection strategy combined with           identification does not require any labeling or immobilization
the inexpensive and HTP experimental methods can lead to               of the test molecule, which makes the approach facile and
the successful functional and structural characterization of           universally applicable. Here, we report a surprisingly simple
protein-protein complexes.                                             version of energetics-based target identification, which only
                                                                       requires ion exchange chromatography, SDS PAGE, and
* These authors equally contributed to this work.                      minimal use of mass spectrometry. The complexity of a
This work was supported by National Institutes of Health               proteome is reduced through prefractionation by ion exchange
Grant GM074942 and by the U.S. Department of Energy,                   chromatography. Urea-induced unfolding of proteins in
Office of Biological and Environmental Research, under                 each fraction is then monitored by pulse proteolysis in the
contract DE-AC02-06CH11357                                             presence and absence of a test molecule. Proteins showing
                                                                       different survival in the presence of the test molecule are
233                                                                    identified by SDS PAGE followed by mass spectrometry. As
                                                                       a proof-of-concept study, we screened E. coli proteome for
Application of a biophysical characterization platform in              ATP-binding proteins. We identified a handful of proteins
developing chemical probes                                             that were not known previously to interact with ATP. We
Guillermo Senisterra, Abdellah Allali Hassani, Alena                   chose phosphoglyceromutase, a glycolytic enzyme whose
Siarheyeva, Gregory Wasney, Irene Chao, Taraneh Hajian,                ATP binding has not been reported, to confirm the proteomics
Peter Brown, Cheryl Arrowsmith, Masoud Vedadi                          screen. Unfolding of recombinant phosphoglyceromutase
Structural Genomics Consortium, University of Toronto,                 indeed was suppressed in the presence of ATP. Our result
Suite 700, 7th Floor, MaRS South Tower, 101 College St.,               demonstrates that the combination of prefractionation and
Toronto, ON, M5G1L5, Canada                                            pulse proteolysis is a simple but powerful way to identify
The advances in large-scale expression and purification                targets of small molecules such as metabolites, allosteric
of recombinant proteins have paved the way for structural              regulators, and drugs.
genomics efforts. Frequently, however, little is known about
newly expressed proteins calling for large-scale protein               235
characterization to better understand their biochemical roles
and to enable structure-function relationship studies. In the          The binding affinity of cyanovirin-N to viral glycoproteins
Structural Genomics Consortium (SGC), we have established              is correlated with the number of functional binding sites
a platform to characterize large numbers of purified proteins.         Irene Maier, Alexandria Berry, Jennifer Keeffe, Sarah
This includes screening for ligands, enzyme assays, peptide            Gillespie, Stephen Mayo
arrays and peptide displacement in a 384-well format.                  Division of Biology, California Institute of Technology, 1200
Here, we describe this platform in more detail and report              E. California Boulevard, Pasadena, CA, 91125
on how this platform coupled with high-resolution X-ray                Transmembrane-linked glycoproteins on the surface of
crystallography and structure-guided methods, can be used              enveloped viruses such as influenza, Ebola, and human


immunodeficiency virus (HIV) initiate viral attachment and               motifs on Sos1. The stoichiometry of the complex between
fusion to host membranes. These trimeric glycoproteins carry             these two molecules is not well understood. In this work, we
the epitopic structures recognized by neutralizing antibodies            investigate the formation of Grb2:Sos1 complexes at multiple
and are thus the primary targets for the design of anti-viral            resolutions. First, we examine the formation of a complex
vaccines and viral entry inhibitors. Their glycosylation also            between the individual polyproline peptides of Sos1 and
makes them an ideal target for carbohydrate-binding proteins             SH3 domains of Grb2 using a combination of evolutionary
such as cyanovirin-N (CV-N), a cyanobacterial lectin known               information and binding energy calculations. Secondly,
for its broad neutralization ability. CV-N’s anti-viral activity         we use a hybrid approach involving molecular dynamics
has been attributed to two distinct carbohydrate-binding sites           simulations and polymer models to estimate the enhancement
that selectively bind N-linked high-mannose oligosaccharides,            in local concentration and intramolecular binding constant of
one with low affinity, and one with high affinity.                       a polyproline motif on Sos1 near the second SH3 domain of
We used surface plasmon resonance and ELISA assays                       Grb2 after the first binding site is occupied. Finally, we use
to measure the binding affinity of CV-N to several viral                 thermodynamic modeling to calculate the stoichiometry and
glycoproteins, including gp120 (HIV), hemagglutinin                      predict the distribution of the complexes that are formed at
(influenza), and Ebola GP1,2 (Ebola). The glycoproteins                  physiological concentrations of the signaling proteins. This
were made recombinantly and expressed in either insect                   is the first such systematic analysis involving the synergistic
or mammalian cells. To determine the effect of avidity on                combination of sequence, structure, and dynamic analyses
binding affinity, studies were done comparing the wild-type              used in order to determine the stoichiometry of the complexes
CV-N monomer (2 binding sites), an engineered variant                    that are dominant in the cellular environment.
that is stable as a domain-swapped dimer (4 binding sites),
and variants designed to knock out 1, 2, or 3 carbohydrate               237
binding sites. All glycoproteins bound with Kds in the nM                Small Molecule Inhibitor Studies with the Human
range, and binding was the same whether expressed in insect              Cytoplasmic Protein Tyrosine Phosphatase and Structural
or mammalian cells. Most importantly, a direct correlation               Elucidation of its Protein Target, EphA2
was observed between binding affinity and the number of                  Greg Costakes1, Kristoff Homan1, Deepa Balasubramaniam1,
functional binding sites (determined using an anti-viral                 Olaf Wiest2, Paul Helquist3, Cynthia Stauffacher1,4
activity assay). Both binding sites of CV-N are able to                  1
                                                                          Biological Sciences, Purdue University, 240 S. Martin
interact with glycoprotein-ligands. The presence of a single             Jischke Drive, West Lafayette, IN, 47907, 2Notre Dame
high-affinity binding site was sufficient to retain binding in           University, 278 SHCB, Notre Dame, IN, 46556, 3Notre Dame
the CV-N monomer, but interestingly, was not adequate in the             University, 361 Stepan, Notre Dame, IN, 46556, 4Purdue
domain-swapped dimer knockouts.                                          University Center for Cancer Research, 201 S. University
                                                                         Street, West Lafayette, IN, 47907
                                                                         EphA2 is a receptor tyrosine kinase that is of particular interest
Interplay between reaction stoichiometry and effective                   because it has been shown to be overexpressed in a number of
concentration: a structure-based synergetic study on                     highly metastatic cancers including breast and prostrate. Non-
Grb2-Sos1 Complex                                                        transformed epithelial cells express basal levels of EphA2
Anurag Sethi1,2, Byron Goldstein1, S Gnanakaran1                         that quickly becomes hyper-phosphorylated and degraded at
  Theoretical Biology and Biophysics, 2Center for Nonlinear              sites of cell-cell contact. In contrast, EphA2 in transformed
Studies, Los Alamos National Lab, Los Alamos, NM, 87545                  epithelial cells is overexpressed, hypo-phosphorylated, and
Multivalent binding, that are formed by the combination                  localized in membrane ruffles. The human cytoplasmic
of relatively weak promiscuous interactions, is a strategy               protein tyrosine phosphatase (HCPTP) is proposed to be
that is often used to increase the affinity and specificity              largely responsible for EphA2 dephosphorylation, and down
of the biomolecular complexes formed by signaling                        regulation of HCPTP results in elevated phosphorylation of
proteins. However, there remains no clear understanding                  EphA2 which reverts cells to a non-transformed phenotype.
of the structural details of how the stoichiometry and the               Docking studies with HCPTP have given insights into
distribution of these complexes are affected by the presence             possible small molecule inhibitors, however no structural
of multivalent interactions in both partners of these signaling          information about their interactions is currently available.
proteins. Growth receptor bound protein-2 (Grb2) is an                   Recent experiments suggest a secondary binding site on
adaptor protein that mediates the recruitment of the nucleotide          HCPTP that may contribute to binding specificity. In order
exchange factor Son of Sevenless-1 (Sos1) from the cytosol               to better understand the interaction between EphA2 and
to the plasma membrane where it activates Ras by inducing                HCPTP, crystallization of the entire cytoplasmic region of
the exchange of GDP for GTP. Grb2 is composed of two SH3                 EphA2 and co-crystallization with HCPTP experiments
domains that can form complexes with multiple polyproline                are currently underway using EphA2 constructs that are
                                                                         specifically designed to aid in crystallization, and optimized


for yield and purity. The structures will provide insight into           with similar rates. The rate of dephosphorylation of these
the dephosphorylation mechanism of EphA2 and also give                   tyrosines is fastest for Y772 with HCPTP-A. In contrast
valuable information needed to develop strong inhibitors for             HCPTP-B appears to prefer Y594 which shows the fastest
HCPTP. Overall these structures will aid in our understanding            rate of dephosphorylation with this isoform. The SAM
of cancer progression within epithelial tissues by determining           domain tyrosine (Y960) is dephosphorylated exclusively
the role of EphA2 phosphorylation with respect to EphA2                  by HCPTP-B. This difference in dephosphorylation sites on
overexpression and cellular localization.                                EphA2 may explain the different roles of the two isoforms of
                                                                         the phosphatase in EphA2 signaling.
Quantitative measurement of dephosphorylation of
EphA2 by both isoforms of the HCPTP using SRM mass                       Coupled folding and binding of the MLL translocation
spectrometry                                                             partners AF4 and AF9 is required for transcriptional
Deepa Balasubramaniam1, Lake Paul2, Kristoff Homan1,                     elongation in acute leukemia
Mark Hall3,4, Cynthia Stauffacher1,4                                     Benjamin Leach1, Tomasz Cierpicki2, Nancy Zeleznik-Le3,
  Biological Sciences, Purdue University, 240 S. Martin Jischke          John Bushweller1
Drive, West Lafayette, IN, 47907, 2Bindley Bioscience Center,            1
                                                                          Molecular physiology and Biological physics, University
Purdue University, 1203 W. State Street, West Lafayette, IN,             of Virginia, rm 4233 Jordan Hall, 1300 Jefferson Park Ave,
47907, 3Biochemistry, Purdue University, 175 S. University               Charlottesville, VA, 22908, 2Department of Pathology,
St., West Lafayette, IN, 47907, 4Purdue Center for Cancer                University of Michigan, 1150 West Medical Center Dr, MSRB
Research, Purdue University, 201 S. University street, West              1, room 4510C, Ann Arbor, MI, 48109, 3Oncology Institute,
Lafayette, IN, 47907                                                     Loyola University Medical Center, 2160 South First Avenue,
Over-expression of the EphA2 receptor tyrosine kinase has been           Maywood, IL, 60153
implicated in aberrant cell signaling in a variety of epithelial         Chromosomal translocations that fuse the Multiple lineage
cancers such as lung, breast and prostate. In transformed                leukemia (MLL) gene in-frame to one of a large number of
cells EphA2 is over-expressed, leading to clustering of the              partners are responsible for a subset of acute leukemias with
receptors at the plasma membrane with a significant decrease             poor prognosis. Although the fusion partners are numerous
in the EphA2 phosphotyrosine content. This decrease in the               and diverse, the two most common, AF4 and AF9, account
phosphotyrosine content concomitant with the increased                   for about 59% of cases between them and have been shown to
protein levels leads to abnormal downstream signaling                    interact in vivo and in vitro. Interestingly, the AF9 C-terminal
events involved in cell migration, proliferation, growth and             domain has been proposed to mediate interactions not only
cytoskeletal rearrangement. The decrease in phosphotyrosine              with AF4 family members, which are involved in regulation
content of EphA2 in different grades of cancer is proposed to            of transcriptional elongation and alternative splicing, but also
be due to decreased contact with its ligand, ephrin A1, as well          the H3K79 methyltransferase Dot1L, human polycomb 3,
as dephosphorylation by the low molecular weight tyrosine                and the BCL6 corepressor. It was previously demonstrated
phosphatases, HCPTP-A and/or B. The overexpression of                    that disruption of the AF4-AF9 interaction using synthetic
HCPTP in non-transformed breast cell lines can promote                   peptides was sufficient to cause necrotic cell death in MLL-
tumorigenesis. This promotion of tumorigenesis by HCPTP                  AF4 cell lines, however the lack of structural data for these
has been linked to the dephosphorylation of EphA2.                       proteins has precluded any efforts at drug development
However, the exact sites of dephosphorylation by HCPTP-A                 targeting this interaction. Using NMR spectroscopy and
and HCPTP-B on the EphA2 receptor are not known.                         other biophysical methods we have shown that this domain
To evaluate which sites on the receptor are dephosphorylated             is intrinsically disordered in solution and upon binding to
by HCPTP-A and HCPTP-B, time-resolved selected reaction                  AF4 these proteins fold synergistically to form a novel mixed
monitoring (SRM) mass spectrometry was employed using                    alpha-beta structure. This interaction is critical for their role
label-free quantification of phosphorylation stoichiometry               in transcriptional elongation and oncogenesis. Alternative
and dephosphorylation rates. This series of experiments                  synergistic folding may explain how the AF9 C-terminal
revealed a preference for different phosphorylation sites                domain can mediate interactions with numerous proteins with
by HCPTP-A and HCPTP-B. Our results suggest that both                    different biological consequences for hox gene regulation in
isoforms can dephosphorylate the EphA2 receptor although                 normal tissues and malignancies.
the rate and specificity of dephosphorylation for specific               The work was supported by The Leukemia and Lymphoma
tyrosines are different. The activation loop tyrosine (Y772)             Society SCOR grant 7006-05 (to J.H.B.).
is dephosphorylated substantially faster by HCPTP-A
than HCPTP-B. The juxtamembrane tyrosines (Y575,
Y588 and Y594) are dephosphorylated by both isoforms


240                                                                       due to intermolecular disulfide bonds formation via cysteine
Understanding carbohydrate recognition by the antiviral
lectin cyanovirin-N                                                       In the present study, we replaced the four cysteine residues
Yukiji Fujimoto1, David Green2,3                                          with alanine in Hpn to abolish disulfide bond formation.
  Chemistry, Stony Brook University, 104 Chemistry, Stony                 Growth assay showed that Escherichia coli BL21 (DE3)
Brook, NY, 11794-3400, 2Applied Mathematics and Statistics,               transformed with mutant hpn gene can still protect the
  Graduate Program in Biochemistry & Structural Biology,                  cells from nickel tolerance, but with lower OD values. This
Stony Brook University, Math Tower, Room 1-117, Stony                     result implies that the mutated protein has lower nickel-
Brook, NY, 11794-3600                                                     binding capacity as the wild type hpn in vivo. The mutant
                                                                          protein was over-expressed and purified. We show that the
The initial recognition of target cells by the human                      mutated protein still aggregates, and aggregation state is in
immunodeficiency virus (HIV) is mediated by the envelope                  equilibrium. Therefore, the underlying mechanism of hpn
glycoprotein, gp120; gp120 is heavily glycosylated, being                 protein aggregation may not only result from intermolecular
nearly 50% carbohydrate by mass. This provides an                         disulfide bond formation, but also from the interactions
interesting opportunity for the development of novel antiviral            among other amino acids, such as histidines.
agents that act through interactions with the viral glycans,
and carbohydrate-binding proteins from a number of natural                We also studied the interactions between UreE and Hpn/Hpnl.
sources have been shown to have potent antiviral activities.              UreE is one of the accessory proteins for urease maturation
While several of these have been quite well characterized                 and is believed to facilitate the insertion of nickel into apo
both structurally and biochemically, there remain many open               urease. UreE binds 0.5 nickel per monomer, this capacity
questions regarding their mechanism of inhibition. To begin               increase to 2.5 when a his-rich tail artificially added. Our
to answer some of these questions, we have applied a diverse              hypothesis is that UreE may interact with Hpn and Hpnl
range of computational approaches to provide insight into the             through the six conserved histidine residues.
structural and energetic determinants of specific oligosaccharide         In the present study, our Co-IP result have provided evidence
recognition by one of the best studied of the virucidal lectins,          that UreE interacts with Hpn and Hpnl. Interestingly, the
cyanovirin-N (CVN). Large-scale molecular dynamics                        interaction between UreE and Hpnl is enhanced by nickel,
simulations have identified important structural features                 i.e., nickel-containing Hpnl has a stronger interaction with
that are difficult to resolve experimentally; and binding free            UreE. This is consistent with the predicted nickel-insertion
energies of a diverse set of oligosaccharide targets computed             role of UreE in urease maturation.
from these structural ensembles give remarkable agreement                 Keywords: Hpn, Hpn-like, histidine-rich protein, Helicobacter
with experiment. Detailed decompositions of the binding                   pylori, nickel, aggregation, UreE, interaction.
free energies on a residue-by-residue basis have additionally
identified several key interactions that define broad affinity            242
for α-1,2-dimannose-containing sugars, as well as a number
of determinants of specificity. The computational integration             Micropatterning of plasma membrane proteins to analyze
of structural studies on representative oligosaccharides                  raft localization in living cells
has additionally allowed us to explore the feasibility of                 Stefan Sunzenauer1, Julian Weghuber1, Mario Brameshuber1,
multivalent interactions. Taken together, these data provide              Lawrence Rajendran2, Gerhard Schuetz1
significant insight into the mechanism of inhibitory activity.            1
                                                                           Institute of Biophysics, JKU Linz, Altenbergerstraße
                                                                          69, Linz, A-4040, Austria, 2Systems and Cell Biology of
241                                                                       Neurodegeneration, University of Zurich, August-Forel Str.
                                                                          1, Zurich, 8008, Switzerland
Expression and purification of mutated hpn protein, and
                                                                          We have developed an assay for quantitative analysis of the
studies of interaction between UreE and Hpn/Hpnl
                                                                          interaction between a fluorescently marked protein (prey)
Shuang Qi
                                                                          and a membrane protein (bait) using microstructured surfaces
Biochemistry, The University of Hong Kong, 302 Laboratory
                                                                          covered with biotinylated ligands (antibodies) targeted
Block, LKS Faculty of Medicine, 21 Sassoon Road, Pokfulam,
                                                                          against the bait. The proof-of-concept was demonstrated for
Hong Kong, Hong Kong, 00852, China
                                                                          the interaction between CD4, a major co-receptor in T-cell
Hpn and Hpn-like are two small nickel-storage proteins in                 signalling, and Lck, a protein tyrosine kinase essential for
Helicobacter pylori. Consisting of 60 amino acids, Hpn is                 early T cell signalling. Here we present improvements
rich in histidine (28 histidine residues), glutamate, glycine             and a more precise characterization of the method as well
and serine residues. There are also four cysteine residues                as the applicability of the assay for the analysis of protein
that may play important roles in its structure and function.              interactions within lipid rafts in the inner and outer leaflet
Our previous study shows that Hpn exists in equilibrium                   of the plasma membrane. We stably expressed fluorescently
among different multimeric forms in solution, which may be


labelled raft and non-raft proteins in the human T24 cell line         docking benchmark set (by Weng’s group) show that our
as prey proteins and determined the degree of interaction              closeness score significantly improves docking performance
with the antibody-targeted bait proteins CD59 (GPI-anchored            for both ZDOCK 2.3 and ZDOCK 3.0. It also suggests that
protein, raft marker) and CD71 (Transferrin-receptor, non-             the local minima distant from the global one can contribute to
raft marker), respectively. We found strong interaction of             make general slope toward native complex which guide rapid
CD59 with putative raft markers including various GPI-GFP              protein-protein association.
constructs, the inner-leaflet associated protein Flottilin1-
GFP, a Pleckstrin-Homology domain fused to GFP and weak                244
interaction with Actin-GFP. Importantly, we did not find
interaction of CD59 with CD71-GFP and other potential                  Investigation of Thrombin Interactions withABE-I Ligands
non-raft proteins. When CD71 was used as the bait protein              using HDX and MALDI-TOF Mass Spectrometry
we did not find interaction with the putative raft markers.            Marina Malovichko, Muriel Maurer
While the detected absence of CD71 from and the presence               Chemistry, University of Louisville, 2320 South Brook St.,
of CD59 in lipid rafts confirm current knowledge, it is still          Louisville, KY, 40292
very unclear if a lipid-raft dependent coupling of proteins            The blood coagulation enzyme thrombin can be triggered
and certain especially negatively charged lipids across the            either to procoagulation/anticoagulation or platelet activation,
plasma-membrane bilayer exists. Thus, our micropatterning              depending on what ligand binds to it. Two highly positive
assay will be of great interest to address this question.              patches termed anion binding exosites ABE I and ABE II
                                                                       aid binding and alignment of substrates for their optimal
243                                                                    interaction with the thrombin active site. In the current
                                                                       work, we focused on the thrombin ligands hirudin (54-65),
Re-scoring protein-protein docking decoys by                           PAR1 (47-62), and PAR3 (44-56). Earlier X-ray crystal
characterizing energy landscape                                        structures and mutant studies proved that these ligands target
Keunwan Park, Dongsup Kim                                              ABE I of thrombin, but the mechanisms of binding were not
KAIST, 373-1, Guseong-dong, Yuseong-gu, Daejeon, Korea,                completely revealed. Hydrogen deuterium exchange (HDX)
Republic of (South)                                                    coupled with MALDI-TOF mass spectrometry provided
The protein-protein interactions require a rapid and highly            an opportunity to map sites of contacts and probe for local
specific association process which leads to appropriate                and long range conformational effects. As anticipated, all
encounter along the complex energy landscape. Long-range               three ligands hirudin (54-65), PAR1 (47-62) and PAR3 (44-
electrostatic and/or desolvation forces have been known to             56) protected the ABE I (65-84) fragment of thrombin from
accelerate the global search to the near-native conformation           deuteration. In addition, we observed some protection of
efficiently by forming a transient encounter complex.                  ABE II (85-99 and 173-181) fragments by PAR3 (44-56).
Therefore, due to this steered diffusion, the energy landscape         For hirudin (54-65), the protection of ABE II fragments was
around the near-native conformation may have distinctive               even smaller. The protection at ABE II was completely lost
features different to random. Under the assumption, we                 for both ligands at 10 minutes of deuteration. Strikingly,
develop the re-scoring method for thousands of docking                 PAR1 (47-62) had more influence on ABE II and remained
decoys by characterizing energy landscape (e.g. width,                 ABE II protected even at 10 minute deuteration. To test if
depth and ruggness). More specifically, 3,600 docking                  occupying the active site can influence the ABE I binding
conformations generated from ZDOCK 2.3 or 3.0 software                 events, we introduced PPACK prior to binding ligands of
are represented by a specific network whose nodes are                  interest. PPACK inhibition of thrombin further increased
cluster-centers (<2.5 Å interface C-alpha RMSD) of docking             protection of ABE II in the presence of PAR3 (44-56) but did
conformations and edges are connected depending on mutual              not influence the hirudin (54-65) dependent ABE II events.
distance. The edge is weighted by the docking scores (and              The results obtained support long range communication
their difference) of adjacent nodes. The directed connection           between two anion binding exosites. The observed effects can
from i to j node means that the two docking conformations              vary from ligand to ligand and may be further influenced by
(nodes) are physically-neighboring, and the high edge-weight           occupying the active site. These solution based studies help
represents that conformational transition from i to j tends to         to better understand the complexity of thrombin regulation.
be occurred very easily. Using the network representation,             (NIH HL068440)
the node satisfying low free energy (high depth), wide and
smooth energy landscape near it is detected by closeness
score around N Å distance. The closeness score of a node is
calculated by summing all shortest path lengths going into
the node, so the nodes which have lower free energy, wider
(many neighbors) and smoother energy landscape would
have higher scores. The results applied to the protein-protein


245                                                                     of pupil constriction, suppression of melatonin synthesis,
                                                                        and direct photic regulation of sleep. Despite its location
Acyl-homoserine lactone induces a conformational change                 in the vertebrate retina, the sequence of melanopsin places
of the quorum sensing transcriptional regulator SdiA                    it phylogenetically with the invertebrate photopigments.
Maria Gallardo1, Nicolas Guiliani1, Peter Jr. Prevelige2,               This raises the question of whether it functions more like a
Octavio Monasterio1                                                     vertebrate or invertebrate photopigment. The light induced
  Laboratorio de Biología Estructural y Molecular,                      depolarization of ipRGCs also extremely elongated. The
Departamento Biología, Universidad de Chile, Las Palmeras               general model of GPCR deactivation has phosphorylation
3425, Ñuñoa, Santiago, Chile, 2Dept. of Microbiology,                   of the carboxy terminal tail by a member of the G-protein
University of Alabama at Birmingham, 845 19th St. South,                coupled receptor kinase (GRK) family which both inhibits
Birmingham, AL, 35294-2170                                              further G protein activation and serves as a signal for the
SdiA is a quorum-sensing transcriptional regulator that                 binding of arrestin which completely quenches the response.
controls the expression of the ftsQAZ operon in bacterial               We are investigating if melanopsin uses this stereotypical
cell division. Like other quorum sensing proteins, SdiA                 deactivation mechanism, or if its long deactive kinetics is
comprises two functional domains. The C-terminal domain                 due a different mechanism. Here we show that melanopsin
contains a predicted helix-turn-helix motif implicated in DNA           heterologously expressed in HEK 293 cells is phosphorylated
binding, and the N-terminal domain binds the autoinducer, .             in a light dependent manner and that this phosphorylation is
In previous work we expressed SdiA protein in the presence              functionally relevant in the deactivation response. Through
of the autoinducer molecule and observed an improvement in              cross-linking and co-immunoprecipitation we demonstrate a
protein solubility. Thus with C8-HSL the SdiA protein was               direct interaction between GRK2 and GRK3 with melanopsin
found in higher quantities in the soluble fraction. Otherwise,          in HEK 293 cells. We also show that phosphorylation inhibits
in the control experiment, the protein was found mainly in the          G protein activation and is required for high affinity binding
pellet. We infer that the autoinducer molecule is important in          of arrestin.
the folding process of SdiA protein. To examine the effect
of the AHL molecule on the protein structure hydrogen                   247
exchange combined with mass spectrometry was used. This
method allows the analysis of the structure and dynamics of             Phosphorylation status of kinases and its implications in
proteins. In this study, the H/D exchange profile of SdiA was           drug discovery
monitored by mass spectrometry to identify the structural               Kallol Ray, Jacquelyn Sikora, James Yu, Bo-Sheng Pan
difference between the protein with or without AHL.                     In Vitro Sciences, Merck Research Laboratories, 33 Avenue
Significant exchange protection was seen in the N-terminal              Louis Pasteur, Boston, MA, 02115
domain in the presence of the autoinducer. A similar degree             Kinases play essential roles in many physiological systems,
of protection was observed in the DNA binding domain of the             making them attractive drug targets. The binding of ATP and
SdiA protein when AHL was added. This study demonstrated                phosphorylation of the kinase promotes a conformational
that the AHL binding to SdiA induce a conformational change             change that activates enzyme activity and creates recognition
in the N-terminal domain that also alters the DNA binding               sites for substrates. Many kinases share highly conserved
domain. FONDECYT 1095121.                                               active sites, therefore identifying specific inhibitors can
                                                                        be a challenge. However, unphosphorylated kinases adopt
                                                                        structurally different conformations, allowing the possibility
Receptors, Signaling and Signal                                         for allosteric inhibition by small molecules. Such inhibitors
Transduction (246 – 261)                                                may bind to the inactive form and prevent further activation.
                                                                        Therefore, the use of unphosphorylated kinases can have a
                                                                        major impact on finding both ATP-competitive and novel non-
                                                                        ATP-competitive inhibitors. Here we have summarized some
Phosphorylation Dependent Deactivation of Melanopsin                    of the case studies highlighting the role of phosphorylation in
Joseph Blasic, Phyllis Robinson                                         such kinase targets.
University of Maryland Baltimore County, 1000 Hilltop
Circle, Baltimore, MD, 21250
Melanopsin is the most recently discovered opsin
photoreceptor in the vertebrate retina. Melanopsin is expressed
exclusively in a small subset of retinal ganglion cells (RGCs)
termed intrinsically photosensitive (ipRGC). In mammals,
these cells have been implicated in non-image forming
functions including regulation of circadian rhythms, control


248                                                                    249
Structural Basis of Shh Regulation by Neutralizing                     Dissecting    Phototaxis    in  the     Cyanobacterium
Antibody 5E1 and Hedgehog-Interacting Protein: Role of                 Synechocystis sp. Strain PCC6803
the Shh Pseudo-Active Site in Signaling                                Susanne Wisén1, Doron Levy2, Devaki Bhaya1
Henry Maun1, Ivan Bosanac2, Suzie J. Scales3, Xiaohui                  1
                                                                        Department of Plant Biology, Carnegie Institution for
Wen3, Andreas Lingel1, J. Fernando Bazan1, Frederic J. de              Sciences, Stanford, 260 Panama St., Stanford, CA, 94305,
Sauvage3, Sarah G. Hymowitz2, Robert A. Lazarus1                       2
                                                                        Department of Mathematics and CSCAMM, University of
  Department of Protein Engineering, 2Department of Structural         Maryland, 4146 CSIC Building #406, Paint Branch Drive,
Biology, 3Department of Molecular Biology, Genentech, Inc.,            College Park, MD, 20742-3289
1 DNA Way, South San Francisco, CA, 94080                              Many photosynthetic organisms, like cyanobacteria, live in
Secreted morphogens Sonic hedgehog (Shh), Indian hedgehog              environments with major fluctuations in vital inputs such as
and Desert hedgehog are important for proper cellular                  nutrients and light. In order to adjust for the differences in
differentiation during embryogenesis. While Hh signaling               light, some species have evolved the ability to quickly sense
is mostly quiescent in adults, dysregulation of this pathway           and respond to differences in both light quality and intensity.
has been implicated in cancer, where inhibition shows                  The unicellular cyanobacterium Synechocystis PCC6803
therapeutic benefit. Although Shh contains a tetrahedrally             exhibits phototaxis – the capability of moving along a
coordinated Zn2+ cation and has structural similarity to Zn2+          surface towards light. This process requires Type IV pili
metalloproteases, it is a pseudo-protease and enzymatically            and a phytochrome-like photoreceptor coupled to a complex
inactive. In vertebrates, Hh ligand binding to Patched1                signal transduction pathway. In a screen for mutants with an
(Ptc1), a 12-pass transmembrane protein, relieves inhibition           abnormal response to light, we uncovered several gene loci
of the 7-pass transmembrane protein Smoothened (Smo),                  that play a role in phototaxis, including genes involved in
leading to activation of Gli transcription factors and target          pilus synthesis, potential photoreceptors, and chaperones.
gene expression. The Shh-neutralizing antibody 5E1 and                 Here we describe the characteristics of some of the mutants,
receptor Hedgehog-interacting protein (Hhip) are potent                and what roles these genes play in the intricate relationship
inhibitors of Hh signaling. We solved crystal structures of            between light response and phototaxis.
chimeric 5E1(ch5E1) Fab and Hhip, each complexed with                  We found that the motility of Synechocystis is a result of a
Shh, which revealed a common binding site on Shh, but                  complex and dynamic interplay between the characteristics
different binding modes. Hhip binds to the pseudo-active               of an individual bacterium and the social dynamics between
site groove on Shh via three loops from its β-propeller                cells. The ability to move over a surface is highly dependent
domain, one of which directly coordinates the Zn2+ cation,             on a well functioning pilus apparatus. This includes both
whereas ch5E1 blocks access to pseudo-active site, but                 the physical ability of the pilus to adhere to the surface and
without Zn2+ coordination. We characterized the inhibitory             retract to generate movement, as well as the capability of pili
activity of ch5E1 and various Hhip ectodomains in cell-                to make contact with other cells, strongly indicating that the
based assays as well as their binding affinities in vitro and          interaction between cells facilitates motility. A number of
show that both have maximal activity when Zn2+ and Ca2+                genes were found to be under control of the cAMP receptor
are bound to Shh. Unexpectedly, sequence comparisons of                protein and dependent on cAMP levels. Inactivation of these
the main Shh-binding loop in Hhip with Ptc1 ectodomains                genes resulted in non- or back-movers. In addition, we are
suggested that Ptc1 also binds to the pseudo-active site. We           using mathematical modeling to investigate wildtype and
confirmed this by NMR binding studies with Hhip- and Ptc1-             mutant motility.
derived peptides and also found that Hhip or ch5E1 compete
Shh binding to Ptc1 over-expressed on cells. Importantly,              Conclusions: We have uncovered new genes involved in
mutations in the pseudo-active site on Indian hedgehog are             phototaxis in Synechocystis PCC6803 and revealed their
linked to Brachydactyly type A1, an autosomal dominant                 function, giving us further insight in the complex signal
inherited disease. Thus, the Shh pseudo-catalytic site plays           transduction networks that control the fascinating process of
an important role in receptor interaction and signaling, where         phototaxis.
targeting may have therapeutic value.


250                                                                      signaling pathway. To address whether Gtr1-Gtr2 complex
                                                                         formation is indispensable for their function, we introduce
Withdrawn                                                                point mutations into both Gtr1and Gtr2 to examine the
                                                                         importance of these amino acid residues in their function. By
251                                                                      motif search, we found that possible leucine zipper motifs,
Genetically engineered scFv against stage specific                       which involved in dimer formation in many proteins, are
embryonic antigens promote isolation of pluripotent bone                 present in both Gtr1p and Gtr2p around the same region
marrow stem cells                                                        of these proteins. Yeast strains harboring mutation in these
Marek Malecki1, Bianka Malecki2                                          leucine zipper motifs failed to complement Gtr1p or Gtr2p
 Western University, 309 E. Second Street, Pomona, CA, 91766-            mutation, suggesting that these motifs are important for
1854, 2PBMEC, 252 Montana, San Francisco, CA, 94112                      dimer formation and biologic function. We found that Kog1,
                                                                         a component of TOR complex, interacts with Gtr1 in yeast
Regenerative medicine is emerging as one of the most                     two hybrid assay. We propose that Gtr1-Gtr2 complex plays
revolutionary fields of the 21st century. Omnipotent stem cells          roles in TOR signal transduction pathway through Kog1
(SC) carry a great potential for becoming the chief medicines
of the medicine of the century. Recent discoveries have                  253
validated omnipotency of adult stem cells. Small populations
of SC residing in bone marrow (BMSC) have been found to                  Enhanced Expression of Cation Channel of Sperm
express certain omnipotent markers such as Stage Specific                (CatSper) in GC-1 spg Cells by Ginseng Radix
Embryonic Antigens (SSEAs), Oct4 and Nanog. In the past,                 Treatment
we genetically engineered single chain variable fragments                Eun Hwa Park, Mun Seog Chang, Seong Kyu Park, Woong
(scFv) against these BMSC markers. Using these scFv, we                  Mo Yang
were able to identify omnipotent BMSC in situ. One of                    Department of Prescriptionology, College of Oriental Medicine,
the fundamental problems for clinical trials of autologous               Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu,
transplants is the difficulty in isolation and propagation of            Seoul, 130-701, Korea, Republic of (South)
BMSC as well as their monitoring upon transplantation. The               Ginseng Radix has been used for the treatment of male
ultimate goal of this work is to develop probes, which will              reproductive problems in traditional Korean medicine. The
allow us to screen and select for omnipotent BMSC providing              Ca2+ channels residing in sperm have led to the recent cloning
a new standard for stem cell purification. Ultimately, this will         and characterization of a novel gene, named CatSper(Cation
facilitate the routes for autologous, stem cell transplants for          channel of sperm). CatSper family (CatSper1-4) proteins are
regenerative therapy. Via controlled differentiation, which              putative ion channels expressed exclusively in membrane of
will eliminate possibilities of cancerous transformation,                the testis and sperm flagellum. It plays an essential role in
genetically engineered tissue will replace deteriorated organs           sperm motility, penetration into the oocyte, and ultimately in
due to injuries, diseases, and side effects of therapies.                male fertility. In this study, the relationship between Ginseng
                                                                         Radix and its effects associated with CatSper genes expression
252                                                                      and spermatogonia proliferation has been examined in vitro.
Role of Gtr1-Gtr2 complex formation on their biologic                    CatSper expressions were assessed by Western blot assay.
function                                                                 There was significantly enhanced expression of CatSper-
Takeshi Sekiguchi1, Yoshiaki Kamada2, Yoshinori Ohsumi3,                 immunoreactive bands in the Ginseng Radix treated group
Yonggang Wang4, Hideki Kobayashi5                                        compared to the vehicle treated group (p < 0.05). Ginseng
  Molecular Biology, Graduate school of medical sciences,                Radix has an enhancing effect of CatSper expression in GC-1
Kyushu University, maidashi3-1-1, Higashi-ku, Fukuoka,                   spg cells at protein level. These findings suggest that Ginseng
812-8582, Japan, 2National Institute for Basic Biology,                  Radix may have a role of improving male infertility related
Nishigonaka 38, Myodaiji, Okazaki, 444-8585, Japan,                      with the sperm alteration and proliferation of germ cells
  Tokyo Institute of Technology, 4259-S2-12 Nagatsuta-cho,               closely related with CatSper gene expression.
Midori-ku, Yokohama, 226-8503, Japan, 4NIH, 18 Library
Drive, MSC-543, Bldg18T, Rm106, Bethesda, MD, 20892,
  Okayama University, tushimanaka2-1-1, Okayama, Japan
Gtr1 and Gtr2 of Saccharomyces cerevisiae are members
of the Ras-like GTP binding family and interact genetically
with Prp20 (yeast RCC1). Gtr1 and Gtr2 were suggested to
be molecular switches in the TOR signaling pathway, which
senses nutrient conditions such as nitrogen level. It remains
unclear how Gtr1 - Gtr2 complex play roles in the TOR


254                                                                   255
Interaction of 18-methoxycoronaridine with nicotinic                  Understanding the molecular basis of Protein-Peptide
acetylcholine receptors in different conformational states            interaction by the two tandem Protein Tyrosine
Katharine Dinwiddie1, Avraham Rosenberg3, Dominik                     Phosphatase domains of Drosophila RPTPs DLAR and
Feuerbach4, Katarzyna Targowska-Duda5, Kryzysztof                     DPTP99A
Jozwiak6, Stanley Glick7, Ruin Moaddel3, Irving Wainer3,              Lalima Madan1, S. Veeranna1, K. Shameer2, R. Sowdhamini2,
Hugo Arias2                                                           B. Gopal1
  Midwestern University, 19555 N. 59th Avenue, Glendale,              1
                                                                       Indian Institute of Science, Lab 301, Molecular Biophysics
AZ, 85308, 2Pharmaceutical Sciences, College of Pharmacy,             Unit, Indian Institute of Science, Bangalore 560012, India,
Midwestern University, 19555 N. 59th Avenue, Glendale,                Bangalore, India, 2National Centre for Biological Sciences,
AZ, 85308, 3Gerontology Research Center, National Institute           National Centre for Biological Sciences,TIFR, GKVK
of Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD,                Campus, Bellary road, Bangalore 560065, Bangalore, India
21224, 4Neuroscience Research, Novartis Institutes for                The Receptor protein tyrosine phosphatases (RPTPs) from
Biomedical Research, Novartis International AG, CH-4002,              the fruit fly Drosophila melanogaster form a good model
Basel, Switzerland, 5Department of Chemistry, 6Clinical               system to examine specific aspects of substrate specificity
Neuroengineering Center, Medical University of Lublin,                as well as different modes of phosphatase regulation. The
Aleja Raclawickie 1, 20-059, Lublin, Poland, 7Center for              catalytic domains of these membrane bound receptors
Neuropharmacology and Neuroscience, Albany Medical                    dephosphorylate the tyrosine residues on their substrate
College, 47 New Scotland Avenue, MC 136, Albany, NY,                  proteins hence modulating key signal transduction pathway(s)
12208                                                                 in Drosophila development. In the present study, we examine
The interaction of 18-methoxycoronaridine (18-MC) with                the substrate specificity of the tandem PTP domains of DLAR
nicotinic acetylcholine receptors (AChRs) was compared                and DPTP99A using tyrosine phosphorylated peptides, with
with that for ibogaine and phencyclidine (PCP). The results           a focus on the role of the second (membrane distal) D2
established that 18-MC: (a) is more potent than ibogaine and          domain in regulation of their catalytic activity. Both in vitro
PCP inhibiting (±)-epibatidine-induced AChR Ca2+ influx.              (Surface Plasmon Resonance and dephosphorylation assays)
The potency of 18-MC is increased after longer pre-incubation         and in silico (Peptide identification and analysis) studies of
periods, which is in agreement with the enhancement of [3H]           these PTP domain-peptide interactions suggests that the D2
cytisine binding to resting but activatable Torpedo AChRs,            domain binds to substrate peptides much tighter than the
(b) binds to a single site in the Torpedo AChR with high              first (membrane proximal) D1 domain and hence sequesters
affinity and inhibits [3H]TCP binding to desensitized AChRs           the peptide from the active domain in the case of DLAR.
in a steric fashion, suggesting the existence of overlapping          D2 thus appears to play an inhibitory role towards D1. In
sites. This is supported by our docking results indicating            the case of DPTP99A, however, the D2 domain binds the
that 18-MC interacts with a domain located between the                peptides with a much lower efficiency as compared to its
serine (position 6’) and valine (position 13’) rings, and (c)         D1 domain. The presence of the D2 domain is however
inhibits [3H]TCP, [3H]ibogaine, and [3H]18-MC binding                 crucial for the enhanced activity of DPTP99A, as seen
to desensitized AChRs with higher affinity compared to                when the two domains are present together. Our studies
resting AChRs. This can be partially attributed to a slower           suggest a clear demarcation in the sequence specificities of
dissociation rate from the desensitized AChR compared to              the D1 and D2 domains of both DLAR and DPTP99A, and
that from the resting AChR. The enthalpic contribution is             that, it is their presence in tandem which decides the final
more important than the entropic contribution when 18-MC              substrate specificities of these Receptors. Small molecule
binds to the desensitized AChR compared to that for the               binding studies using Fluorescence spectroscopy highlight
resting AChR, and vice versa. Ibogaine analogs inhibit the            the differences in the phosphotyrosine binding pockets of
AChR by interacting with a luminal domain that is shared              the two domains of DLAR and DPTP99A. Our studies thus
with PCP, and by inducing desensitization.                            reveal the role of silent D2 domains which may have evolved
Support: Science Foundation Arizona                                   to create a balance between peptide-binding versus peptide-
                                                                      dephosphorylation for this class of proteins.


256                                                                    binding partners of PDZ domains are found mostly in plasma
                                                                       membrane proteins, whereas low to medium affinity binders
Taiwan banded krait protease inhibitor-like protein-1-                 are cytoplasmic or nuclear proteins. Our results explain that
induced apoptosis of human leukemia U937 cells through                 PDZ domain-mediated scaffolding of cell surface receptors is
ADAM17/Lyn/Akt pathways                                                mainly mediated by strong PDZ domain-ligand interactions,
Wen-Hsin Liu, Long-Sen Chang                                           while the assembly of cytoplasmic signaling complexes is
Institute of Biomedical Sciences, National Sun Yat-Sen                 mediated by weak PDZ domain-ligand interactions which
University, No.70, Lianhai Rd., Kaohsiung, Taiwan                      need the help of other domain-domain interactions. Human
     Bungarus multicinctus (Taiwan banded krait) protease              PDZ domain-ligand interaction network is the first network of
inhibitor-like protein-1 (PILP-1) induced apoptosis of human           its kind to provide a systems view of PDZ domain mediated
leukemia U937 cells, characteristic of loss of mitochondrial           cell signaling processes.
membrane potential, degradation of procaspase-8 and
production of t-Bid. FADD down-regulation neither restored             258
viability of PILP-1-treated cells nor attenuated production of
active caspase-8 and t-Bid in PILP-1-treated cells, suggesting         Computational models provide insights into heterotrimeric
that death receptor-mediated pathway was not involved in the           G-protein function
cytotoxicity of PILP-1. It was found that PILP-1-evoked p38            Noel Carrascal1, David Green1,2
MAPK activation and ERK inactivation led to cell death and
                                                                        Department of Applied Mathematics & Statistics, Stony
down-regulation of ADAM17 in U937 cells. Knockdown                     Brook University, Math Tower, Stony Brook, NY, 11794-
of ADAM17 by siRNA induced death of U937 cells and                     3600, 2Graduate Program in Biochemistry and Structural
inactivation of Lyn and Akt. Overexpression of ADAM17,                 Biology, Stony Brook University, Life Science Building,
LynY507F (gain of function) and constitutively active Akt              Stony Brook, NY, 11794
suppressed the cytotoxic effects of PILP-1. PILP-1-elicited            Many of steps in cellular signaling pathways involve specific
inactivation of Lyn and Akt was abrogated in cells with                interactions between proteins in which conformational changes
overexpressed ADAM17 and LynY507F, respectively. Taken                 due to ligand binding or post-translational modification are
together, our data suggest that suppression of ADAM17-                 coupled to complex formation or dissociation. Understanding
mediated Lyn/Akt pathway elucidates the underlying                     the mechanisms by which such specific, conformationally-
mechanism responsible for PILP-1-induced apoptosis of                  regulated interactions are made is fundamental to the
U937 cells.                                                            understanding of cellular signal transduction as a whole.
                                                                       We are beginning to address these questions in the context
257                                                                    of G-protein-coupled-receptor signal transduction, with
                                                                       an initial focus on the coupling of structural dynamics and
Systematic analysis of human PDZ domain-ligand                         energetics to association of the subunits of the heterotrimeric
interaction network and their localization                             G-proteins themselves.
Jinho Kim1, Jae-seong Yang2, Solip Park2, Yoon Sup Choi2,
Jouhyun Jeon1, Jihye Hwang1, Sanguk Kim1,3                             Computational methods provide a powerful approach to
  Division of Molecular and Life Science, 2School of                   this problem, complementary to experimental studies.
Interdisciplinary Bioscience and Bioengineering, 3Division             Molecular dynamics simulations provide details of motions
of IT Convergence Engineering, POSTECH, San 31, Hyoja-                 of the proteins at the atomistic level; structural analysis
dong, Pohang, 790-784, Korea, Republic of (South)                      of these simulations provides an intuitive insight into
                                                                       mechanism. Continuum electrostatic models can further be
PDZ domains play an important role on controlling the                  used to an energetic dimension to this analysis, providing a
signal flow by assembling multiple signaling components,               decomposition of individual contributions to protein stability
such as enzymes, receptors, and ion channels. Elucidation              and complex affinity on a residue-by-residue basis. We have
of PDZ domain interaction network is limited because only              performed such analyses on multiple states of a representative
a small portion of PDZ domain-ligand interactions has been             G-protein — considering both the complexed trimer and the
known. To understand the organization of PDZ domain-                   dissociated Gα monomer and Gβγ dimer as well as both GTP-
ligand interaction network in human genome, we integrated              and GDP-bound states — using dynamic simulations of each
the human protein interactome, the subcellular localizations           system over an extended period of time as a starting point.
of proteins, and the PDZ domain-peptide interactions from              The calculations provide several important insights into the
protein arrays and phage display. The resulting network                mechanism of function in this system. Among these, we find
visualizes the bipartite relationships between human PDZ               that the dynamic ensembles of GTP- and GDP-bound (active
domains and their partners with binding strengths and                  and inactive) Gα overlap, and thus these states are more
subcellular locations of the interactions. From the analyses           similar than suggested by crystallography. Additionally, we
of the network, we discovered the important clue to elucidate          are able to identify sets of residues that play essential roles in
the logic of PDZ domain-mediated scaffolding. High affinity


many aspects of function, from discrimination of nucleoside               of external Ca2+. The ability to monitor and characterize
di- and triphosphosphates by the Gα subunit to the formation              underlying Ca2+ changes will assist in understanding which
of specific Gαβγ complexes.                                               Ca2+ signals cause certain physiological and pathological
                                                                          responses. The underlying Ca2+ signal in all of these
259                                                                       responses is regulated both spatially and temporally by a
                                                                          range of Ca2+-binding proteins. Ca2+ buffer proteins such as
Conformation and activity of the intracellular domain of                  parvalbumin B (PAV) are commonly known to modulate the
the Arabidopsis CRINKLY4 receptor-like kinase                             magnitude and duration of Ca2+ transients in the cytoplasm.
Matthew Meyer1, A. Gururaj Rao2                                           While PAV can further act as an intracellular Ca2+ store,
  Biochemistry, Biophysics, and Molecular Biology, Iowa                   this effect in modulating the Ca2+ signal originating from
State University, 0049 Roy J. Carver Co-labs, Iowa State                  predominant intracellular Ca2+ stores such as the ER is
University, Ames, IA, 50011, 2Biochemistry, Biophysics, and               largely unexplored. The objective of this study was to use
Molecular Biology, Iowa State University, 1210 Molecular                  fluorescence live-cell imaging to monitor Ca2+ changes in
Biology Bldg., Iowa State University, Ames, IA, 50011                     cells expressing cytoplasmic or plasma membrane localized
Arabidopsis CRINKL4 (ACR4) is a serine/threonine receptor-                PAV upon purinergic P2Y receptor or acetylcholine channel
like kinase (RLK) involved in the overall development of the              receptor stimulation. This system allowed us to study the
plant. ACR4 promotes the proper formation of epidermis in                 buffering effects of PAV on Ca2+ signals originating from
both reproductive and vegetative tissues and regulates stem               the ER. We report that the overexpression of PAV delays
cell differentiation in the root meristem. The intracellular              Ca2+-induced Ca2+-release (CICR) from the ER. PAV also
domain (ICD) of the RLK consists of a juxtamembrane                       disrupts the signaling involved in triggering Ca2+ influx from
domain (J), a kinase domain (K) and a carboxy-terminal                    the extracellular medium, suggesting a temporal sensitivity
domain (C). In our laboratory we have undertaken a detailed               between ER depletion and subsequent activation of store-
in vitro characterization of the structure-function relationships         operated Ca2+ entry (SOCE). This work was supported
of the intracellular domain to complement in vivo plant                   by fellowships from the Natural Science and Engineering
studies. To this end, we have recombinantly expressed the                 Research Council (NSERC) and grants from the Canadian
ICD as a soluble, C-terminal fusion to the N utilization                  Institutes of Health Research (CIHR).
substance A (NusA) protein. The fusion protein was purified
by metal affinity chromatography followed by gel filtration.              261
The enzymatic and conformational properties of the ICD
have been characterized. We have determined that (a) the                  Periplasmic sensing of Cu(I)/Ag(I) by CusS
ICD undergoes autophosphorylation via an intramolecular                   Swapna Aravind1, Christopher Rensing2, Megan M.
mechanism, (b) the kinase has the ability to phosphorylate                McEvoy3
exogenous substrates such as myelin basic protein and the
                                                                            Chemistry and Biochemistry, Univeristy of Arizona, 1041 E
NusA tag itself, (c) the ICD undergoes a conformational                   Lowell st, Rm# 509, Bio Sciences West, Tucson, AZ, 85721,
change upon autophosphorylation, and (d) the ICD has the
                                                                            Soil, Water and Environmental Sciences, University of
ability to oligomerize in the absence of phosphorylation.                 Arizona, 1177 E 4th St, Rm# 429, Shantz 38, Tucson, AZ,
                                                                          85721, 3Chemistry and Biochemistry, Univeristy of Arizona,
260                                                                       1041 E Lowell st, Rm# 516, Bio Sciences West, Tucson, AZ,
Over-Expression of Calcium Buffer Protein Delays                          Transition metal homeostasis in cells is maintained by highly
Calcium Signaling and Disrupts Store-Operated Calcium                     regulated uptake, distribution and efflux systems to prevent
Entry                                                                     the lethal effects of high metal concentrations. Copper is
Elizabeth Pham1, Evan Mills1, Kevin Truong1,2                             essential for many biological functions, but in excess can be
  Institute of Biomaterials and Biomedical Engineering,                   highly toxic. Silver does not have a biological function and
University of Toronto, 164 College Street Rm 407,                         is toxic to cells even at low concentrations. Anaerobic copper
Rosebrugh Bldg, IBBME, UofT, Toronto, ON, M5S3G9,                         and silver resistance in Escherichia coli is controlled by a two
Canada, 2Edward S. Rogers Sr. Department of Electrical and                – component regulatory system CusRS that senses metal stress
Computer Engineering, University of Toronto, 10 King’s                    and responds by upregulating the cusCFBA genes. This system
College Circle, Toronto, ON, M5S3G4, Canada                               is activated by CusS, the sensor histidine kinase that initiates
Ca2+ signaling is important for many cellular processes                   the cus response by sensing elevated copper/silver levels in
such as differentiation, proliferation, apoptosis and muscle              the periplasmic space of the bacterium. CusS is anchored to
contraction, to name a few. Elevations of cytosolic Ca2+                  the cytoplasmic membrane of E. coli by two transmembrane
often come from intracellular stores, predominantly from the              domains followed by a cytoplasmic transmitter domain.
endoplasmic reticulum (ER). However, internal Ca2+ stores                 The transmembrane domains are separated by a periplasmic
are finite, so prolonged Ca2+ signaling relies on an influx               region of approximately 150 amino acids. CusS is thought


to sense Cu(I)/Ag(I) via its periplasmic region CusS39-186.                264
Upon metal sensing, CusS transmits the signal to its cognate
response regulator via phosphorylation. Despite its central                Investigations on Conformational Heterogeneity of
role in the Cus system, little is known about the molecular                Ubiquitin by spFRET
mechanisms of CusS sensing.                                                Li-Ling Yang1, Michael, W.-P. Kao2, Hsin-Liang Chen2,
                                                                           Tsong-Shin Lim3, Wunshain Fann4, Rita, P.-Y. Chen2
In this study, we are investigating the structural characteristics         1
                                                                             Institute of Physics, Academia Sinica, 128 Sec. 2, Academia
and metal binding properties of the periplasmic region of CusS.            Rd., Nankang, Taipei, 11529, Taiwan, 2Institute of Biological
The gene for CusS39-186 was cloned into an overexpression                  Chemistry, Academia Sinica, Institute of Biological Chemistry,
vector and the protein was purified from cell extracts. CD                 Academia Sinica, 128 Sec. 2, Academia Rd., Nankang,
spectra for CusS39-186 indicate both alpha helical and beta-               Taipei, 11529, Taiwan, 3Physics, Tunghai University, NO.
strand character. Analytical ultra centrifugation data show                181, SECTION 3, TAICHUNG PORT ROAD, Taichen City,
that the protein is monomeric in solution. These features                  Taichung, 40704, Taiwan, 4Institute of Atomic and Molecular
do not change upon Cu(I)/Ag(I) addition. Initial isothermal                Sciences, Academia Sinica, No. 1, Roosevelt Rd., Sec. 4,
titration calorimetry data show metal binding with micro-                  Taipei, 10617, Taiwan
molar affinity. Further understanding of the actual nature of
the signal and the mechanism of signal detection by CusS will              The role of structural ensembles of ubiquitin in substrate-
contribute to a better understanding of signal transduction in             binding has received a lot of attentions. We try to address
the Cus system.                                                            this issue via single-molecule techniques combining with
                                                                           FRET (Förster Resonance Energy Transfer). FRET serves as
Source of Research Support: NIHGM079192                                    a distance ruler in close proximity (~ 1 nm to ~ 10 nm), and
                                                                           the conformational dynamics of ubiquitin could be observed.
Single Molecule Studies (263 – 269)                                        Two dye molecules, Alexa Fluor (donor) and Alexa Flour 594
                                                                           (acceptor), were specifically labeled onto the ends of β1 and
                                                                           β5 strand. According to the fluorescence intensity of donor
263                                                                        and acceptor, FRET efficiencies which are related to the
Dynamic bending and cleavage of a DNA gate by human                        structure of ubiquitin were determined. Three distinguished
topoisomerase IIα                                                          populations were observed in our FRET efficiency distribution:
Sungchul Hohng1, Sangwha Lee1, Neil Osheroff2                              high FRET (~90%), moderately high FRET (~70%), and low
  Seoul National University, 599 Gwanak-ro, Gwanak-gu,                     FRET (~20%). The transitions between different populations
Seoul, Korea, Republic of (South), 2Biochemistry, Vanderbilt               were recorded as well. The β1-strand was expected to have
University, Department of Biochemistry Vanderbilt University               dynamic deviation in equilibrium to assist the recognition of
School of Medicine 37232-0146, Nashville, TN, 37232                        binding substrates; the labeling sites on the ends of β1 and
The finding of a phenomenon of topology simplification by                  β5 strand would reflect the structural variation. We surmise
ATP-driven topoisomerases, so called the “Rybenkov effect”,                that the decreased compactness and stability of double-dye-
raised an intriguing question: how does an enzyme of less                  labeled ubiquitin molecules might enhance the structural
than 10-nm in diameter recognizes the global topology of                   flexibility. The effects of mutation and dye-labeling on the
DNA double helix, which is enormously larger than the                      dynamic conformational fluctuations are still less explored,
enzyme itself? Among several models proposed to explain                    and more work and efforts are essential to this field.
the phenomenon, the enzyme-induced sharp bending of a
DNA gate is most consistent with experimental evidences to                 265
date. However the dynamic nature of the DNA bending has                    Selective RNA annealing and unwinding mechanisms of
never been directly observed until now.                                    Hfq revealed by single-molecule measurements
In this work, we used single-molecule fluorescence assays                  Wonsok Hwang1, Veronique Arluison3, Sungchul Hohng2
to monitor the binding/dissociation of human topoisomerase                 1
                                                                             Physics and Astronomy, 2Physics and Astronomy, Department
IIá, and accompanying deformation, and cleavage of DNA                     of Biophysics and Chemical Biology, Seoul National
duplexes. As a result, we clearly observed sequential events               University, San 56-1, Gwanakro Sillim-dong, Gwanak-gu,
of enzyme binding, DNA bending, and DNA cleavage. The                      Seoul, 151-747, Korea, Republic of (South), 3Department
sharp bending of DNA duplexes was sequence-specific, and                   of Biophysics and Chemical Biology, University Paris 7,
occurred before DNA cleavage, suggesting that the cleavage                 Laboratoire Leon Brillouin CEA Saclay Bat 563, Paris,
site is selected by the deformability of DNA, not by the                   91191, France
sequence itself. The clamping of the ATP binding domains                   Hfq is a global regulator involved in many post-
increased both magnitude and portion of DNA bending and                    transcriptional regulation processes of prokaryotes. Previous
cleavage, proving the existence of an efficient communication              studies illustrated the close relation of the RNA chaperoning
mechanism of enzyme domain motion and DNA transaction.                     activity of Hfq and its regulatory function—as an example, it


facilitates annealing of DsrA (a small non-coding RNA of E.             267
coli) to rpoS (a regulatory target of DsrA), and also induces
melting of a translation-inhibitory RNA secondary structure             Untying knots in proteins
in rpoS. However the detailed mechanisms of intermolecular              Joanna Sulkowska1, Piotr Sulkowski2, Piotr Szymczak3,
base-paring and strand unwinding induced by Hfq binding are             Jose Onuchic4, Marek Cieplak5
not completely understood yet. In this study, we used single-
                                                                         Center for Theoretical Biological Physics, University of
molecule fluorescence assays to observe individual events of            California San Diego, 7226 Urey Hall, MC0374, 9500
Hfq-mediated RNA annealing/unwinding process by using                   Gilman Dr., La Jolla, CA, 92093, 2physics, Caltech, 1200
RNA fragments from DsrA and rpoS. Our results show that                 E. California Blvd., 452-48, Pasadena, 0, 91125, 3Faculty of
Hfq stimulates the intermolecular base-paring of DsrA and               Physic, Institute of Theoretical Physics, Warsaw University,
rpoS by simultaneously binding both RNAs using same RNA                 Hoza 69, Warsaw, 00-681, Poland, 4Center for Theoretical
binding site on its proximal side and the competition for the           Biological Physics, University of California San Diego,
same binding site of the two RNAs is a necessary step for               7226 Urey Hall, MC0374, 9500 Gilman Dr., La, CA, 92093,
efficient annealing. The binding of Hfq to rpoS partially melts
                                                                         physics, Polish Academy of Science, Al. Lotnikow 32/48,
the inhibitory secondary structure of rpoS, and the complete            02-668 Warszawa, Poland, Warsaw, Poland
melting quickly follows due to the reduced thermal stability            A shoelace can be readily united by pulling by its ends rather
of the structure caused by Hfq binding.                                 than by its loop.
Keywords: Hfq, rpoS, DsrA, single-molecule FRET                         Attempting to untie a native knot or slipknot my results in
This work was supported by Creative Research Initiatives                an outcome which is much less unique since its intrinsic
(Physical Genetics Laboratory, 2009-0081562) of National                structure has non-uniform mechanical properties. We used
Research Foundation of Korea                                            structure based model to shown that:
                                                                        1. stretching slipknots reveals a surprising growth of
266                                                                        their unfolding times when the stretching force crosses
                                                                           an intermediate threshold. This behavior arises as a
Unfolding and Refolding Dynamics of Filamin A Protein                      consequence of intermediate states in which the slipknot is
under Constant Forces                                                      jammed, and which correspond to so-called catch bonds.
Hu Chen1, Xiaoying Zhu2, Fumihiko Nakamura3, Michael P.                 2. pulling knotted proteins by specific amino acids may cause
Sheetz1,4, Jie Yan1,2                                                      retraction of a terminal segment of the backbone from
  RCE in Mechanobiology, T-Lab, 5A Engineering Drive                       the knotting loop and untangle the knot, as opposite to
1, Singapore, Singapore, 2Physics, National University                     pulling by termini. At still other amino acids, the outcome
of Singapore, 2 Science Drive 3, Singapore, Singapore,                     of pulling can go either way. We study the dependence of
  Department of Medicine, Harvard Medical School, Brigham                  the untying probability on the way the protein is grasped,
& Women’s Hospital, Boston, MA, 02115, 4Department of                      the pulling speed, v, and the temperature, T. Elucidation
Biological Sciences, Columbia University, 713 Fairchild                    of the mechanisms underlying this dependence is critical
Center, M.C. 2408, New York, NY, 10027                                     for a successful experimental realization of protein knot
For the first time, the dynamical response of two segments                 untying.
of Fimalin A protein, 1-8 domains and 16-23 domains,
to constant stretching forces was investigated by using                 268
magnetic tweezers. For FLNA 1-8, Stepwise unfolding was
found at ~50 pN, while stepwise refolding was found when                Engineering Cysteine Residues into Glyoxysomal Malate
force dropped to below 10 pN. Refolding often took tens of              Dehydrogenase to Study Subunit Interactions Using
seconds to a few minutes. Fluctuation between unfolded state            Fluorescence and Atomic Force Microscopy Approaches
and a more compact state was found before folding to the                MJ Drake, Ellis Bell
stable, presumably the native state. Our results suggest that           Chemistry, University of Richmond, Gottwald Science
Filamin A refolds through intermediate states. For FLNA 16-             Center, University of Richmond, 28 Westhampton Way,
23, some structures can be disrupted by force ~10 pN only,              Richmond, VA, 23173
which indicates the interactions between domains can be                 Despite the fact that Malate Dehydrogenase was one of the
opened under physiologically relevant forces to expose the              first multisubunit enzymes to have its structure determined
binding site which is blocked without stretching force.                 by x ray crystallography many questions remain about
                                                                        the mechanism and role of subunit interactions in this
                                                                        protein. To provide a quantitative framework to analyze
                                                                        engineered interface mutants, a single cysteine residue has
                                                                        been engineered into the subunit on the outer edge, furthest
                                                                        from the interface and shown to be reactive [the five native


cysteine residues all appear buried and inactive in the native             type heat shock: at elevated temperatures mGFP-GPI homo-
form of the enzyme]. Random labeling of the 2 cysteines per                association disappeared, accompanied by an increase in the
dimer with Alexa Fluor 488/AF647 [Ro approximately 52A]                    expression of the small heat shock protein Hsp27. Beside the
or AF546/AF647 [Ro approximately 63A] is being used to                     detection of this indirect, lipid-mediated protein association
allow resonance energy transfer measurements between the                   our TOCCSL-technique allows for the observation of direct
two halves of the dimer to probe ligand induced changes over               homo- and hetero-association of mobile proteins in the live
long distances and effects on dimer stability. In an alternative           cell plasma membrane or in model systems.
approach preliminary data has been obtained focused on using               1 Moertelmaier, M., Brameshuber, M., Linimeier, M.,
the engineered sulfhydryl groups to immobilize the dimer on                  Schütz, G. J. & Stockinger, H. Thinning out clusters while
the gold tip and stage of an atomic force microscope to allow                conserving stoichiometry of labeling. Appl Phys Lett 87,
pulling type experiments to probe the effects of ligands on the              263903 (2005).
stability of the dimer interface relative to the protein structure
using analysis of the resultant force curves.
This work is supported by NSF Grant MCB 0448905 to EB                      Bioinformatics, Genomics,
                                                                           Proteomics (270 – 275)
Imaging of mobile stable lipid-mediated protein                            270
associations in the live cell plasma membrane                              Application of Graph-Theoretical Approach and
Mario Brameshuber1, Verena Ruprecht1, Julian Weghuber1,                    Nonlinear Dynamics to Protein Structure Representation,
Imre Gombos2, Ibolya Horvath2, Laszlo Vigh2, Hannes                        Analysis and Prediction
Stockinger3, Gerhard Schuetz1                                              Meyer Pesenson1, Alexander Kister2, Isaac Pesenson3
  Institute for Biophysics, University of Linz, Altenbergerstr.            1
                                                                             IPAC, Caltech, m/c 220-6, Pasadena, CA, 91125, 2Dept. of
69, Linz, 4040, Austria, 2Biological Research Center,                      Mathematics, Rutgers University, 110 Frelinghuysen Rd.,
Hungarian Academy of Sciences, P.O. Box 521, Szeged,                       Piscatawa, 08854, 3Dept. of Mathematics, Temple University,
6701, Hungary, 3Department of Molecular Immunology,                        1801 N. Broad Street, Philadelphia, PA, 19122
Medical University of Vienna, Lazarettgasse 19, Vienna,
1090, Austria                                                              Methods for the comparison of biological sequences
                                                                           constitute a very important part of bioinformatics. Direct
The plasma membrane has been hypothesized to contain                       approaches to protein structure determination such as
nanoscopic lipid platforms, also termed lipid or membrane                  X-ray crystallography and nuclear magnetic resonance are
rafts. Based on biochemical and cell biological studies,                   expensive, time-consuming and not always applicable.
rafts are believed to play a crucial role in many signaling                Because of this, the gap between known sequences and solved
processes. However, there is currently not much information                structures is quickly widening. In this paper we describe a
on their size, shape, stability, surface density, composition and          theoretical approach to protein structure prediction as well
heterogeneity. We present here a method which allows for the               as to general bio-information representation and analysis.
first time the demonstration that single rafts diffuse as stable           The starting point of our approach is the well-known use of
platforms in the live cell plasma membrane. Our method                     graph-theoretical methods applied to graphs representing the
senses rafts by their property to assemble a characteristic                structure of proteins. First we develop harmonic analysis on
set of fluorescent marker-proteins or lipids on a time-scale               graphs and manifolds. Furthermore, we consider networks
of seconds. The special photobleaching protocol TOCCSL                     whose nodes represent the dynamical units and study their
(Thinning Out Clusters while Conserving Stoichiometry of                   nonlinear collective dynamics. These developments enable us
Labeling1) was used to reduce the surface density of labeled               to describe systems’ structural properties, as well as model the
mobile rafts down to the level of well-isolated diffraction-               effects that these structural properties have on the dynamical
limited spots, without altering the single spot brightness.                processes running in networks. Our approaches complement
The statistical distribution of probe molecules per raft was               each other and besides their obvious theoretical interest will
determined by single molecule brightness analysis. For                     have practical implications by enabling analysis of biological
demonstration, we used the consensus markers Bodipy-GM1, a                 organization principles at different scales.
fluorescent lipid analogue, and glycosylphosphatidylinositol-
anchored monomeric GFP. For both markers we found
cholesterol-dependent association in the plasma membrane
of living CHO and Jurkat T cells in the resting state,
indicating the presence of small, mobile, stable rafts hosting
these probes. We further applied the technology to address
structural changes in the plasma membrane during fever-


271                                                                      272
Pan Phylum Analysis of Protein-Protein Interactions                      Proteomic Analysis of Copper Response in E. coli
Reveals Potential Drug Targets for Helminths                             Alayna George Thompson1, Greggory Longoni2, Megan M
Christina Taylor1, Sahar Abubucker1, Zhengyuan Wang1,                    McEvoy1
John Martin1, Kerstin Fischer2, Peter Fischer2, Dao Jun Jiang2,          1
                                                                          Chemistry and Biochemistry, University of Arizona, 1041 E
Gary Weil2, Makedonka Mitreva1                                           Lowell St, Tucson, AZ, 85712, 2Chemistry and Biochemistry,
  Genetics, Washington University School of Medicine, 4444               University of Arizona, 1041 E Lowell St, Tucson, AZ,
Forest Park Boulevard, St. Louis, MO, 63108, 2Division                   85721
of Infectious Diseases, Washington University School of                  Metal resistant bacteria are a public health threat because of
Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110-1093                  the rising use of metals as biocides. Copper and silver are
Parasitic helminths have deleterious effects on human health,            commonly used in clinical and environmental settings as
livestock, and plants, costing society billions of dollars               biocides, and are the focus of this research. Previous work
annually. Finding new drug targets for parasitic infections              has identified genes and proteins involved in copper and
would be of great utility for humanity, as there is a large need         silver resistance, though a complete understanding of how
to develop new drugs to fight parasitic helminth infections              resistance is achieved is not well understood. The objective
due to the developing resistance and side effects of current             of this study is to quantify the metal-induced expression
treatments.                                                              changes of proteins in E. coli.
This study underlines three major principles: i) proteins                To quantify changes in protein abundance, isotopic labeling
that are essential and conserved among species that span a               was coupled with mass spectrometry (MS). Wild type E. coli
phyla are of greatest value, as they provide foundations for             strain W3110 and derivatives were cultured in media with
developing broad control strategies, ii) Parasite proteins               either natural abundance of nitrogen isotopes, or enriched in the
that share homology to the host counterpart are also of great            15N isotope to differentiate control and metal treated cultures.
value when they posses molecular features that are unique to             At collection, equal amounts of control and treatment culture
the parasite are therefore candidates for selective targeting,           were mixed and centrifuged; harvested cells were lysed and
and iii) proteins rarely act in isolation, and the majority of           applied to a 1D polyacrylamide gel for separation. Protein
biological processes occur via interactions with other proteins,         bands were excised, alkylated, digested, and extracted, and
so protein-protein interactions offer a realm of unexplored              then analyzed using MS/MS. Quantification was measured by
potential drug targets. Here we present a computational                  the relative abundance of heavy and light peptides. To date,
approach which builds on these three principles, utilizing               cells have been cultured and analysis is underway. We are
complete proteomes of the model free-living Caenorhabditis               also using this method to measure translocation of proteins
elegans, 6 parasitic helminths and 2 of their hosts. Markov              to the periplasm.
clustering of the proteins resulted in orthologous families              Funding: Science Foundation Arizona (AMGT), NIH
that could be placed in species specific groups. Protein-                GM079192 (MM)
protein interactions within these species specific groups were
identified by comparison to evidence based protein-protein               273
interactions. Protein-protein interactions specific to nematodes
were prioritized and scored based on RNAi phenotype and                  A novel method for protein-protein interaction site
homology to the PDB. In addition, investigation of the                   prediction with phylogenetic substitution models
parasite protein-protein interactions shared with the host               David La, Daisuke Kihara
resulted in amino acid insertions and deletions specific to the          Department of Biological Sciences, Purdue University,
parasites. Developmental gene expression profiles, functional            Hockmeyer Hall of Structural Biology, Purdue University,
annotation (GO), and druggability were also considered.                  249 South Martin Jischke Drive, West Lafayette, IN, 47907
Several protein-protein interactions unique to nematodes or              Protein-protein binding events mediate many critical
with nematode specific amino acid insertions and deletions               biological functions in the cell. In particular, these processes
emerged from this study and provide novel potential drug                 directly involve functionally important amino acids are
targets for controlling parasitic helminth infections.                   conventionally accepted as conserved throughout evolutionary
                                                                         time. However, protein-protein interaction sites exhibit
                                                                         higher sequence variation than other functional regions, such
                                                                         as those that correspond to catalytic sites and non-protein
                                                                         ligand binding sites. Consequently, protein interfaces
                                                                         that result in such semi-conserved nature pose significant
                                                                         challenges in protein-protein interaction site prediction. To
                                                                         approach this problem, we develop a phylogenetic framework


to capture the mutational behavior of essential protein-protein          275
binding residues. Through the comprehensive analysis of
functionally diverse protein families, we discover key amino             Local sequence hydropathy in folded proteins
acid substitution patterns that are characteristic of protein-           Ian Borukhovich, Matthew H. J. Cordes
protein interfaces. Effectively, we demonstrate the contrast             Chemistry and Biochemistry, University of Arizona, 1041 E.
between interface and non-interface substitution models show             Lowell St., Biosciences West 436, Tucson, AZ, 85721-0088
essential mutational constraints imposed on protein-protein              Protein sequences may experience evolutionary pressure
binding residues. Based on this analysis, we develop a novel             against highly hydrophobic local regions of sequence, in
method, BindML, which utilizes these evolutionary models                 part because such sequences promote nonnative aggregation.
to predict protein-protein binding sites of protein structures           In an attempt to uncover and study trends in local sequence
even without knowledge of their interacting partners. When               hydrophobicity, and in particular to discern any deviations
assessed on a large benchmark of protein complexes, our                  from trends expected from simple native state structural
method generally outperforms alternative methods for                     requirements, we conducted two sequence/structure
protein binding interface prediction. A firm understanding of            analyses of folded single domain proteins in the ASTRAL
the mutation patterns of protein-protein binding sites would             compendium. First, we examined the average hydropathy of
help further unravel the mysteries behind the protein-protein            sequences flanking completely buried residues of all amino
binding phenomena.                                                       acid types. We found that the hydropathy of a buried residue
                                                                         negatively correlates with the average hydropathy of short
274                                                                      sequences flanking that residue. Second, we examined the
                                                                         composition of 6 and 8 residue local and nonlocal sequence
Cysteine Oxidation Prediction Program (COPP): A new                      groups exhibiting overall levels of solvent accessibility
software program that predicts reversible protein cysteine               ranging from 0 to 50%. We found that while both local and
thiol oxidation reactions.                                               nonlocal hydropathy correlate negatively and approximately
Ricardo Sanchez, Larry Grant, Jamil Momand                               linearly with solvent exposure, local sequences show a flatter
Chemistry and Biochemistry, California State University-                 slope with less extreme hydropathy values at both low and
LA, 5151 State University Dr., Los Angeles, CA, 90032                    high solvent exposure. The results of both analyses are
A software program called the Cysteine Oxidation Prediction              consistent with stronger evolutionary constraints on local
Program (COPP) was created and is accessible by internet                 than on global sequence hydropathy.
( through a Graphical User Interface.
COPP uses molecular structure data from the Protein Data Bank
to predict reversible cysteine thiol oxidation susceptibility in         Chromatography and Protein
proteins. COPP was created from an algorithm generated via
the decision tree output of the J48 machine learning program.
                                                                         Purification (276 – 283)
COPP was shown to be 80% accurate by jackknife analysis of
a database that contains 161 oxidation susceptible cysteines
and 161 oxidation non-susceptible cysteines. Of the cysteine             Cloning, Expression, Purification and Functional
thiols in the oxidation-susceptible portion of the database,             Characterization of Recombinant Aleuria aurantia
61% are in intramolecular disulfide bonds to cysteines and               Lectin
39% covalently bind to oxygen, glutathione, or intermolecular            Larry Birkenmeyer, Tracey Rae, Qiaoqiao Ruan, Sam Diep,
protein cysteine residue thiols. Protein S-nitrosylation is              Cheng Zhao, Robert Ziemann, Ryan Bonn, Scott Muerhoff
a common event but predicting target cysteine thiols that                Abbott Laboratories, 100 Abbott Park Rd., Abbott Park, IL,
become S-nitrosylated remains problematic. We used 29                    60064
known sites of S-nitrosylation to test the accuracy of our               Aleuria aurantia lectin (AAL) is a fucose-specific binding
COPP program and found that 20 were predicted correctly,                 lectin originally isolated from the orange peel mushroom. The
giving an accuracy of 69%. Out of the 20 that were predicted             protein is composed of two identical 312 amino acid beta-
accurately, 19 were predicted to occur because the calculated            propeller subunits. Each subunit contains five binding sites
target cysteine thiol pKa was equal to or less than 9.05 and             for fucose. We sought to clone and expressed this protein in E.
there was greater than 1.3 A2 calculated surface exposure of             coli and utilize the purified protein as a reagent for detection
the thiol group.                                                         of fucosylated proteins. The gene was expressed with a
                                                                         carboxyl terminal hexahistidine tag in E. coli BL21(DE3)
                                                                         cells. The rALL was purified to homogeneity from the soluble
                                                                         fraction via immobilized metal affinity chromatography. The
                                                                         pure protein exhibited essentially the same characteristics as
                                                                         native protein based on SDS-PAGE, ESI mass spectrometry,


and circular dichroism. The rAAL and nAAL were essentially              from oxidation damage by binding and sequestering heme.
the same. Labeling of rAAL with biotin and subsequent                   The heme-hemopexin complex is the taken up by hepatocytes
detection of analyte-rALL-biotin complexes using HRPO-                  via a receptor (LRP/CD91) mediated mechanism where heme
streptavidin demonstrated the ability of rALL to bind to                is released and subsequently degraded while hemopexin is
fucosylated proteins. These results demonstrate comparable              recycled into the serum where it can continue to scavenge
biological function of native and recombinant AAL and the               free heme. We sought to clone and express full-length human
utility of the latter for development of fucosylated protein            hemopexin for use in immunoassay development. The
detection assays.                                                       complete human sequence was cloned into pcDNA3.1 and
                                                                        transfected into HEK293 cells growing in suspension culture
277                                                                     in bovine serum-free medium. The inclusion of the natural
                                                                        signal/leader sequence allowed the protein to be secreted into
Bacterial co-expression as a convenient method for the                  the cell culture medium where it was easily detected by SDS-
production and purification of core histones                            PAGE and western blotting using commercially available
Robert Dutnall1,2, Megan Anderson2, Joon Huh3, Thien Ngo2,              monoclonal antibodies. The protein exhibited characteristics
Alice Lee2, Genaro Hernandez4, Joy Pang2, Jennifer Perkins2             similar to that of the native protein purified from human plasma
  Department of Chemistry and Biochemistry, University                  based on circular dichroism spectroscopy, immunoreactivity,
of San Diego, 5998 Alcala Park, San Diego, CA, 92110,                   and ability to bind iron-protoporphyrin. These results showed
  Division of Biological Sciences, 3Department of Chemistry             that the recombinant HPX can substitute for native protein in
and Biochemistry, University of California San Diego, 9500              immunoassays.
Gilman Drive, La Jolla, CA, 92093, 4Department of Biology,
San Diego State University, 5500 Campanile Drive, San                   279
Diego, CA, 92182
Co-expression of proteins in bacteria offers an important               An Amine Activated Agarose support for quick and
strategy for producing multiprotein complexes for                       efficient immobilization of proteins and antibodies
biochemical and biophysical studies. We have found that co-             Ramesh Ganapathy, Mark Schofield, Atul Deshpande,
expression of histones H2A and H2B (from yeast, chicken                 Surbhi Desai
or Drosophila) leads to production of soluble heterodimeric             Research & Development, Thermo Fisher Scientific, 3747 N
H2AH2B complexes. Drosophila histones H3 and H4 can also                Meridian Road, Rockford, IL, 61101
be produced as a soluble (H3H4)2 heterotetrameric complex               Affinity chromatography is an important technique for
if they are co-expressed with the histone chaperone Asf1.               enriching, purifying and identifying proteins and antibodies.
The soluble H2AH2B and (H3H4)2 can be purified by simple                Various reactive chemistries such as amine, sulfhydryl,
chromatographic techniques and have similar properties to               carboxyl and carbohydrate are available for immobilization
endogenous histones. Our methods should facilitate histone              of biomolecules. Aldehyde activated agarose and cyanogen
production for studies of chromatin structure and regulatory            bromide activated agarose are widely used for coupling
proteins that interact with histones. We describe a simple              amine reactive molecules. However, there are limitations
strategy for constructing co-expression plasmids, based on              using these chemistries including the use of toxic sodium
the T7 RNA polymerase system, which is applicable to other              cyanoborohydride, long reaction times and leaching of
systems. It offers several advantages for quickly creating              coupled ligand. Pierce NHS Activated Agarose enables
plasmids to express two or more proteins and for testing                simple and efficient covalent immobilization of proteins at
different combinations of proteins for optimal complex                  pH 7-9 forming stable amide linkages. This resin is easy to
production, solubility or activity.                                     use and requires a one step reaction with minimal hands on
                                                                        time. We have also demonstrated the advantage that our dry
278                                                                     resin provides for coupling proteins to NHS-activated agarose
                                                                        resin at low concentrations.
Cloning, expression and characterization of recombinant
human hemopexin in HEK293 cells                                         We have demonstrated quick (within one hour) and efficient
Bruce Dille1, Larry Birkenmeyer1, Ruthie Coffey1, Qiaoqiao              coupling (≥ 80 %) using NHS Activated Agarose with
Ruan2, Sylvia Saldana2, Sergey Tetin2, Mary Pinkus3, Tracey             different molecular weight proteins, such as Human IgG (mol
Rae4, A. Scott Muerhoff1                                                wt = 150 KDa), Streptavidin (mol wt = 60 kDa) and Green
 Biomolecular Research, 2Molecular Binding Characterization,            Fluorescent Protein (mol wt = 25 kDa). We have also shown
  Antibody Research, 4Analytical Chemistry, Abbott                      superior coupling capacity of NHS Activated Agarose to a
Laboratories, 100 Abbott Park Rd., Abbott Park, IL, 60064               variety of proteins compared to competitor products. We have
                                                                        also demonstrated the performance of our NHS Ester Resin
Hemopexin (HPX) is a 462 amino acid serum protein secreted              in immunoprecipitation applications. Here we immobilized
by liver that exhibits tight binding to iron protoporphyrin (Kd         Anti MAP Kinase and used it to pull down MAP Kinase from
~ 1 pM). The protein participates in the protection of cells            HeLa cell lysate.


280                                                                    opportunity for spectroscopic detection. A fluormetric dye,
                                                                       N-phenyl-1-naphthylamine (NPN), is known to increase the
Estimating protein molecular weight using the extended                 emission quantum yield of luminescence when incorporated
range prestained Precision Plus Protein Dual Xtra                      inside of the micelle. To determine polysorbate 80
Standards                                                              concentration above its CMC, we have developed a single
Joseph Siino, Sansan Lin, Jonathan Kohn, Dennis Yee,                   step HPLC isocratic flow through a knitted reaction coil
Michael Urban                                                          with fluorescence detection. NPN is added into the mobile
Life Science Group, Bio-Rad Laboratories, 6000 James                   phase, as the sample is loaded onto the knitted coil the
Watson Drive, Hercules, CA, 94547                                      number of micelles increases, hence increases the emission
Prestained protein standards are tools commonly used                   of NPN as it partitions inside of the additional micelles. The
with one dimensional electrophoresis to monitor or track               chromatograms display a single peak at the elution point of
electrophoresis. The pattern of bands (or ladder) resulting            polysorbate 80, and the response of this peak is proportional
from the electrophoretic separation a prestained standard              to the concentration of the surfactant in the sample.
can also be used to estimate protein molecular weight. The
relative mobility of the proteins in the standard can be used          282
to make a standard curve from which the molecular weights
of unknown proteins can be estimated. The accuracy of                  Novel Purification Protocol for Heparin Binding Proteins
the estimate depends upon polyacrylamide percentage, gel               Sumit Batra, Nilesh Sahi, Kristen Mikulcik, Heather
chemistry, quality of the prestained protein standards and use         Shockley, Camille Turner, Eric Conte, Rajalingam
of an appropriate model (or function) for the data.                    Dakshinamurthy
                                                                       Chemistry, Western Kentucky University, 1906 College
In this study, the molecular weights of five known sample              Heights Blvd. #11079, Bowling Green, KY, 42101
proteins (ranging from 3.5 to 180 kD) were estimated using
the new Bio-Rad Precision Plus Protein Dual Xtra Standards             Heparin binding proteins regulates a wide range of important
(range 2 to 250 kD) on a variety of gel formulations. Both             cellular processes including angiogenesis, wound repair,
linear and nonlinear functions were used to model the                  survival of neurons, tumours and pathogenesis of various
standard data and then applied to the sample protein data. In          diseases. The functional and biopharmaceutical importance
general, acrylamide gradient gels achieve a wider molecular            of heparin binding proteins and the disadvantages of heparin
weight range compatible with linear regression modeling                affinity chromatography including long experimental time,
of the standard than do single percentage acrylamide gels.             high cost and maintenance of heparin column propel us to
However, nonlinear models of the protein standard can be               devise a new purification protocol for the heparin binding
used effectively on both gradient and single percentage                proteins which are expressed from Escherichia coli. This
acrylamide gel types. The Precision Plus Protein Dual Xtra             novel off column purification method depends on multiple
Standards is a convenient, ready to use prestained standard            amberlite ion exchange resins chromatography. Different
effective for estimating molecular weights over an expanded            resins were used such as IRC 50, CG 50, IRP 64 etc. with
range on a variety of gel chemistries.                                 elution using tris-HCl and phosphate buffers of differing
                                                                       ionic composition and pH. These resins were used because of
281                                                                    their high exchange capacity from the presence of carboxylic
                                                                       acid functional groups. We purified two heparin binding
HPLC Fluorescent Micelle Assay For Accurate                            proteins to homogeneity (~98% pure) using IRC 50 which
Determination of Polysorbate 80 Concentration in                       include human fibroblast growth factor (FGF) which was
Therapeutic Protein Formulations                                       expressed in a soluble form and D2 domain of FGF receptor
Christopher Chen, Anisa Vaidya, Tia Estey, Laura Salmeron,             which was expressed in an insoluble form in inclusion
Joseph Siemiatkoski                                                    bodies. The authenticity of expression and purification were
Biogen Idec, 5200 Research Place, La Jolla, CA, 92122                  checked by SDS-PAGE. The purified proteins human FGF-1
Polysorbate 80 is a nonionic surfactant and emulsifier that is         and D2 domain of FGF receptor were further analyzed by
widely used as an excipient in protein formulations to prevent         steady state fluorescence, Far UV circular dichroism (CD)
protein aggregation and surface adsorption. For therapeutic            and isothermal titration calorimetry (ITC) to understand their
proteins, the addition and correct optimization of polysorbate         structure and function. The results of this study not only pave
80 can help reduce aggregate and particle formation during             way for development of efficient purification protocol for
long-term storage as well as during process-related stress.            heparin binding proteins but also for other cationic proteins
Therefore, it is critical to develop a method to accurately            to understand structure-functional relationship of biological
determine the concentration of polysorbate 80 in various               important biopharmaceuticals.
protein formulations. The ability of polysorbate 80 to form
micelles and incorporate fluormetric dyes has provided an


283                                                                     Computational Biology (284 – 286)
Application of Multi-dimensional Reversed-Phase HPLC
for the Isolation of a Calcitonin-Like Diuretic Hormone                 284
in Tenebrio                                                             Ligand Induced Fit Activation Mechanism of GH5
Derek Jensen1, David A. Schooley2                                       Endoglucanase: A Molecular Dynamics Study
  Biochemistry, University of Nevada, 1664 N. Virginia St MS            Somayesadat Badieyan1, David Bevan2, Mike Zhang3
330, Reno, NV, 89557, 2Biochemistry, University of Nevada,              1
                                                                          Biological Systems Engineering, Virginia Tech, 201 Sietz
1664 N Virginia St MS 330, Reno, NV, 89557                              Hall, Virginia Tech, Blacksburg, VA, 24060, 2Biochemistry,
Urine production in insects is a hormone-driven process                 Virginia Tech, 201 Fralin Institute, Blacksburg, VA, 24060,
mechanistically distinct from vertebrates, thus making water
                                                                          Biological Systems Engineering, Virginia Tech, 210 Sietz
balance an attractive target for the development of specific            Hall, Blacksburg, VA, 24060
pesticides. An early step in understanding insect diuresis is           Clostridium thermocellum has garnered research interest due
to identify effecting ligands. There are three primary classes          to its cellulolytic and ethanologenic abilities, being capable
of diuretic hormones in insects: calcitonin-like, corticotropin         of directly converting cellulosic biomass into ethanol. The
releasing factor-like (CRF), and kinins. The focus of this              structure of C. thermocellum cellulosomal endoglucanase
study is to isolate and characterize a calcitonin-like diuretic         CelC, a member of glycoside hydrolases family 5 (GH5), has
hormone (DH) from mealworm beetle, Tenebrio molitor.                    been determined at 2.15 Å resolution, but a loop structure of
Success in any purification scheme hinges on two                        the enzyme (93-105 residues) is missing. To better understand
requirements: the ability to efficiently remove the desired             the mechanism of 1-4-β-polysaccharides hydrolysis by CelC
ligand from a matrix of impurities and the means to                     and the function of the missing loop in overall stabilization of
monitor activity at each step. High performance liquid                  bounded structure between the enzyme and the substrate, new
chromatography (HPLC) has become an indispensable                       simulations have been conducted using Molecular Dynamics
element in natural ligand isolation. Several modes of HPLC              (MD) of CelC. In MD, a model segment of cellulose has
exist that facilitate separation of analytes according to               been docked in the active site of the enzyme. The resulted
various intrinsic chemical properties. Reversed-phase HPLC,             trajectories of MD are analyzed for conformational changes
however, is by far the most powerful in terms of separation             of in-silica built loop 93-105 and the other loops of CelC
power. A diverse assortment of available reversed-phase                 in complex form to reveal the mechanism of induced fit
supports with wide-ranging mobile phase compatibility                   activation. This study aids to uncover those flexible regions
offers selectivity potential unrivaled by alternate LC modes,           of proteins that can be potentially considered as a target for
such as ion exchange or size-exclusion chromatography.                  stability enhancement by site-directed mutagenesis.
In this study we have isolated and characterized a diuretic
peptide (DH31) from the yellow mealworm, Tenebrio molitor.              285
The ligand was purified to homogeneity using exclusively
                                                                        A Protein Trajectory Reconstruction Strategy from
reversed-phase chromatography. The six-step scheme made
                                                                        FRET-derived Distance Constraints
use of alkyl, cyano, and polymeric stationary phases eluted
                                                                        andrej savol, chakra chennubhotla
with acetonitrile gradients containing trifluoroacetic acid
                                                                        Dept. of Computational Biology, University of Pittsburgh,
or heptafluorobutyric acid as ion-pairing agents. Activity
                                                                        3501 Fifth Avenue, Suite 3064 BST3, Pittsburgh, PA, 15260
throughout the series of steps was tracked with a sensitive
competitive ELISA assay. The amino acid sequence of the                 Critical to almost all biological processes, proteins are
purified peptide was determined by Edman sequencing.                    truly the cell’s functional building blocks. The remarkable
                                                                        specificity and diversity of protein function are consequences
This is only the second calcitonin-like diuretic hormone to
                                                                        of native conformational fluctuations, or protein dynamics, in
be isolated and identified from insect tissue, while several
                                                                        the aqueous cytoplasmic environment. While crystallographic
other calcitonin-like DH sequences have been predicted from
                                                                        and NMR-based methods have dramatically improved our
BLAST searches of genome databases for other species.
                                                                        structural interpretation of proteins, extending that knowledge
                                                                        to predict and describe dynamic conformational processes
                                                                        remains a major open challenge. Merging single molecule
                                                                        Forster Resonance Energy Transfer (smFRET) data from
                                                                        single molecule experiments with Elastic-Network protein
                                                                        Models (ENM), we reconstruct dynamic adenlyate kinase
                                                                        (ADK) trajectories over extended time scales and throughout
                                                                        its catalytic process.


With exceptional temporal resolution, smFRET experiments                Enzyme Kinetics and Mechanism
can determine the efficiency of energy transfer between an
acceptor and donor fluorophore, each a modified cysteine                (287 – 290)
residue, in a single mutated protein. FRET efficiency can
be converted into a time-evolving distance, here treated as             287
a perturbation of the equilibrium distance from the PDB
structure, which is reduced to an elastic network with only             Biochemical Characterization of the Artificial S262T
one alpha-carbon atom at each node. Normal Mode Analysis                Mutant of the Metallo-beta-lactamase IMP-1
(NMA) reveals the natural directions of motion most                     Kevin Pegg, Alex George, Kyu Lee, Peter Oelschlaeger
responsible for the (inter-residue) distance perturbation, and          Chemistry Department, California State Polytechnic
these same eigen modes inform the geometry of all other                 University, Pomona, 3801 W. Temple Ave., Pomona, CA,
(non-tagged) nodes. We use a Kalman-filter based approach               91768
to reconstruct the entire protein network at each FRET time             Metallo-β-lactamases (MBLs) are increasingly important
point. Determining a maximum-likelihood estimation of                   factors of bacterial resistance against β-lactam antibiotics,
the protein trajectory permits spatial and statistical analysis         including carbapenems. The two wild-type MBLs IMP-1 and
of regions both proximal and distal to the tagged residues.             IMP-6 differ by only amino acid residue 262, which is serine
The method easily accepts additional constraints (bond- or              in IMP-1 and glycine in IMP-6. Other residues, including
dihedral-angle) or more detailed covariance matrices from               valine, have been introduced at the same position. In this study,
MD simulations or structural alignments.                                we examine the effect of threonine at this position on enzyme
                                                                        expression, stability, and activity. Our hypothesis is that
286                                                                     the properties of the mutant IMP-1-S262T are intermediate
                                                                        to those of IMP-1 and IMP-1-S262V, because threonine
Investigation of the layered structure of HIV-1 gp120                   exhibits characteristics of both serine (hydroxyl group) and
using temperature accelerated molecular dynamics                        valine (bulky). IMP-1, IMP-1-S262V, and IMP-1-S262T
Ali Emileh, Cameron Abrams                                              were expressed under the control of the T7 promoter in E.
Chemical Engineering, Drexel University, 3141 Chestnut St,              coli. Cells were lysed by sonication and the soluble fractions
Philadelphia, PA, 19104                                                 were separated from the insoluble fractions by centrifugation.
The HIV-1 virus is decorated on the surface by glycoprotein             The solubilities of MBLs were estimated by comparing
spikes. Each spike is a trimer of dimers of gp120 and gp41.             the soluble and insoluble fractions by polyacrylamide gel
gp120 is the component responsible for target receptor                  electrophoresis. The conversion of nitrocefin by the soluble
attachment and fusion initiation. When gp120 binds to the               fractions right after cell lysis and after prolonged incubation
CD4 receptor on the surface of a target cell, it undergoes an           at room temperature and at 4 ºC was used to determine the
unusually large conformational change. This conformational              relative enzyme activity and stability. The amount of soluble
change is thought to lead to exposure of the fusion peptides            protein after recombinant expression, enzyme activity, and
on gp41 which proceed to insert into the target membrane                stability were superior for IMP-1, closely followed by IMP-1-
and through a poorly understood conformational change,                  S262T, and followed with a bigger margin by IMP-1-S262V.
result in membrane fusion. One of the major obstacles in                These results indicate that the properties of IMP-1-S262T are
designing entry inhibitors or immunogens has been the lack              indeed intermediate to those of IMP-1 and IMP-1-S262V.
of a crystal structure of the spike in its unliganded native            IMP-1-S262T would probably confer sufficient antibiotic
form and equivalently, a crystal structure of the unliganded            resistance to bacteria if it occurred naturally.
gp120. Very recently, it was proposed that gp120 is structured          This research was supported by an award from Research
into a layered topology which allows for its very high degree           Corporation for Science Advancement.
of plasticity and also guarantees its function. We used
temperature accelerated molecular dynamics (TAMD) as                    288
a tool to explore the conformational landscape of gp120.
Through this technique we were able to observe in atomistic             Computational approach to investigate the catalytic
details, how the layering mechanism may be implemented                  chemical reaction mechanism of glyoxysomal Malate
by gp120 to hide its conserved inner domain from immune                 Dehydrogenase (gMDH)
surveillance. We discuss the molecular basis for some of the            Hugo Guterres, Ellis Bell
mutational effects observed experimentally in the context of            Chemistry, University of Richmond, Gottwald Science
a layered structure.                                                    Center, University of Richmond, 28 Westhampton Way,
                                                                        Richmond, VA, 23173
                                                                        Geometry optimization calculation is used to find the
                                                                        minimum total energy and structure of the transition state
                                                                        during the conversion of malate to oxaloacetate catalysed by


the enzyme glyoxysomal Malate Dehydrogenase (gMDH).                     of the enzyme which exhibit both negative cooperativity and
During the forward catalytic reaction, the proton and hydride           substrate inhibition, and its allosteric regulation by ADP [an
from malate transfer to the active site of gMDH and cofactor            activator] or GTP [an inhibitor]. The results suggest that both
nicotinamide adenine dinucleotide (NAD+). Our primary goal              crowding effects and simple viscosity effects come into play
is to develop a computational approach to examine the effects           and must be considered when assessing the in vivo roles of
of active site and second sphere mutations in the activity of           allosteric effects in this complex enzyme.
gMDH. Gaussian 09 is used to develop the structure of the               This work is supported by NSF Grant MCB 0448905 to EB
compounds, arrange their symmetry, and run the geometry
optimization calculations. Initially a truncated model that             290
only included only Histidine 220, malate and NAD+ was
built to analyze the effect of active site. The result showed           Identification of Residues in the Propeptide of Pseudomonas
an imaginary frequency which indicates the presence of                  aeruginosa Elastase with Roles in Chaperone Function
the transition state. The geometry structure of the transition          Emily Boice1, Efrat Kessler2, Dennis Ohman1,3
state only confirms the hydride transfer, whereas the proton            1
                                                                         Microbiology, Virginia Commonwealth University, 1101
transfer is disoriented. Since this is a truncated model of the         East Marshall St., Box 980678, Richmond, VA, 23298,
reaction, it suggests that the amino acids around the active            2
                                                                         Tel-Aviv University, Sheba Medical Center, Tel-Hashomer,
site play important role in facilitating the hydride and proton         Israel, 3Veterans Affairs Medical Ctr, 1201 Broad Rock Blvd,
transfer. We are systematically enlarging the qm portion of the         Richmond, VA, 23249
calculations to include more amino acids around the active              Bacterium Pseudomonas aeruginosa secretes several
site to determine which play critical roles. Currently we have          proteases associated with pathogenesis, but the most abundant
finished the transition state energy for the native gMDH and            and active is elastase (M4 metalloendopeptidase). Elastase
are working to find the total energy for transition state of a          is synthesized as a zymogen with propeptide (18-kDa) and
variety of mutants of gMDH. The total activation energy for             mature (33-kDa) domains. Our objective here was to determine
the transition state can be computed and compared between               which conserved residues, across bacterial species, play a
the native gMDH and the mutants gMDH and is being used to               role in the propeptide’s chaperone activity using site-directed
facilitate the study of a variety of “second sphere” mutations          mutagenesis. The propeptide and mature were expressed
around the active site of the enzyme.                                   as separate proteins, which still permitted extracellular
This work is supported by NSF Grant MCB 0448905 to EB                   secretion of active enzyme in Pseudomonas. When secreted
                                                                        elastase activity was tested using a fluorogenic substrate, 19
289                                                                     propeptide mutants had 100-fold higher Km values compared
                                                                        to wild-type, and 9 others showed no elastolytic activity, thus
The effect of molecular crowding on allosteric kinetics                 revealing which residues were critical for secretion of an
and allosteric regulation of Glutamate Dehydrogenase                    active complex. A 3D model was made of the propeptide-
Sam Asante, Ellis Bell                                                  mature complex, and it suggested that E155 was at the
Chemistry, University of Richmond, Gottwald Science                     proteins’ interface, and residues L58, G105 and D114 were on
Center, University of Richmond, 28 Westhampton Way,                     the exterior of the complex. To determine which substitutions
Richmond, VA, 23173                                                     caused a mature-folding defect, propeptides were tested in an
In vivo proteins exist in an environment quite different from           in vitro refolding assay. Purified active elastase and affinity
that usually used in test tube experiments, with total in vivo          tagged recombinant propeptides (expressed in E. coli) were
protein concentration estimated to be in the range of 200-              denatured in guanidine-HCl, mixed and allowed to renature
400mg/mL. Although the impact of such “crowding” on                     together during dialysis. When activity was analyzed, G105
protein structure and function is currently a topic of much             had a Km value close to wild-type, suggesting a role in
research, little attention has been paid to possible effects on         secretion rather than folding. However, E155, K70 and E76
allosteric regulation of function. Glutamate Dehydrogenase,             had Km values 100-fold higher, suggesting roles in folding.
with a plethora of well documented allosteric effects                   An analysis of purified propeptide by circular dichroism
involving both global and local flexibility as well as                  showed evidence for stable secondary structure, which may
intersubunit interactions offers an excellent system to study           be important for folding of mature. Overall these studies
the effects of crowding. In Vivo glutamate dehydrogenase                revealed conserved residues in the propeptide that have
concentrations have been estimated as being approximately               roles in the folding, activation and secretion of the mature
2mg/mL in a background of a total protein concentration                 elastase.
in the mitochonrial matrix of possibly 400mg/mL. We                     Research supported by NIH-NIAID.
have examined the effects of crowding using either inert
proteins such as BSA or viscosity changes produced by small
molecule viscants such as glycerol on the initial rate kinetics


Membrane Proteins (291 – 294)                                            fully inactive structure. Our predicted CB1 protein structures
                                                                         of the wild-type and mutants show conformational changes
                                                                         that suggest a mechanism of activation in this receptor that
                                                                         may be broadly applicable to Class A GPCRs. Our lowest-
A Computational Study of Translocon-Assisted Insertion                   energy wild-type conformation has a conserved ionic lock
of Helical Proteins Into Membranes                                       between R3.50 and D6.30, which has been observed in the
Anna Rychkova, Arieh Warshel                                             crystal structure of bovine rhodopsin. Furthermore, the
Chemistry, University of Southern California, 3620                       important residue T210 forms a hydrogen-bond with S158
McClintock Ave, Los Angeles, CA, 90089-1062                              (2.45). Through activation, the TM3-TM6 ionic lock is
The insertion of transmembrane (TM) proteins into                        broken, freeing the residues to form hydrogen-bonds with
membranes is a subject of great current interest. It is                  other residues. Our lowest-energy T210I mutant has an ionic
known that the recognition of the protein is performed by                lock between R5.71 and D6.30. This ionic lock pattern agrees
the translocons, complex molecular machines which ensure                 with the crystal structure of opsin, thought to be close to the
both the translocation of globular proteins across membranes             active form of rhodopsin. Also, R3.50 is free to interact with
and the integration of membrane proteins into membranes.                 S2.45 in this mutant. The docking results of an agonist and
Biochemical studies have provided major information about                an antagonist to the three receptor forms of CB1 will also be
the insertion process and structural studies have started to             presented.
provide hints about the insertion mechanism and about the
corresponding energetics.                                                293
In the present work we used a coarse grained (CG) model                  NMR Structural Studies of Mercury Transport Membrane
to explore the energetics of the insertion of transmembrane              Proteins
helixes into membranes trough the translocon. It is concluded            Woo Sung Son, George Lu, Henry Nothnagel, Aubrey Davis,
that the apparent insertion free energy of TM with charged               Stanley Opella
residues probably reflects more than just the free energy                Chemistry and Biochemistry, UCSD, 9500 Gilman Drive, La
of moving the isolate single helix into the membrane. The                Jolla, CA, 92093-0307
present approach should be effective not only in exploring the           Exposure to high levels of mercury has severe effects on
mechanism of operation of the tarnslocon but also in providing           human health and negatively impacts local ecologies, therefore
an effective way for extrapolating experimental observations             the development of methods to remediate areas contaminated
into energy based model of membrane proteins.                            with this heavy metal are of interest. In mammals, inorganic
                                                                         mercury ions (Hg2+) have been linked with damage of
292                                                                      several vital organs. Some bacteria, on the other hand, have
GPCR Activation: Structural Insights From the CB1                        been observed to thrive in the presence of Hg2+. This is
Cannabinoid Receptor                                                     accomplished through the presence of the bacterial mercury
Caitlin Scott, Ravinder Abrol, William Goddard III                       detoxification system, which reduces inorganic mercury
Chemistry, Caltech, MC 139-74, Caltech, Pasadena, CA,                    (Hg2+) to the less toxic and volatile atomic mercury (Hg0).
91125                                                                    The expression of Mer proteins in both bacteria and plants
                                                                         has already shown promise in the development of low cost
The cannabinoid CB1 receptor is a G-protein-coupled                      bioremediation solutions; however, further progress could
receptor (GPCR) located in the central nervous system,                   be made in the production of more efficient systems through
which is activated by the psychoactive drug cannabis and                 deeper understanding of the protein’s mechanism of action.
endocannabinoid neurotransmitters. We have predicted the                 Various isolates of the Mer operon encode four membrane
structure of human CB1 wild-type receptor and two mutants                proteins (MerE, MerF, MerT and MerC) that vary in length,
(T210A, T210I) from first principles using the GEnSeMBLE                 helical topology and proposed metal binding motifs. Current
(GPCR Ensemble of Structures in Membrane BiLayer                         research entails the atomic resolution characterization of the
Environment) method. Debra A. Kendall and her co-workers at              four proteins, with the goal of elucidating the mechanism
the University of Connecticut have shown that these mutations            of Hg transport for each. Progress towards determining the
of the Threonine 210 on the third transmembrane helix (3.46              three-dimensional structures of the two two-helix proteins,
position by Ballesteros numbering) can significantly change              MerE and MerF, will be presented.
the receptor activation profile (D’Antona et al., Biochemistry
2006, 45, 5606). They showed that the T210I mutation causes
the protein to become fully active as evidenced by increased
binding affinity for agonists and decreased binding affinity for
antagonists and inverse agonists. The T210A mutated protein
had the opposite profile indicating that the protein adopts a


294                                                                        value decomposition. The analysis assigned the signal to
                                                                           two states whose dynamics clearly differ. Surprisingly, even
Structure and Dynamics of Vpu from HIV-1 by NMR                            at ATP saturation, ~50% of the molecules still populate the
Hua Zhang, Yan Wang, Sang Ho Park, Leong Chan, Stanley                     T state at any instance of time, indicating constant out-of-
Opella                                                                     equilibrium cycling between T and R. Only upon addition of
Chemistry and Biochemistry, UCSD, 9500 Gilman Drive, La                    the co-chaperonin GroES does the T-state population vanish.
Jolla, CA, 92093-0307
                                                                           Our results suggest a model in which the T/R ratio is controlled
The viroporin protein Vpu is an 81-residue membrane                        by the rate of ADP release after hydrolysis, which can be as
protein encoded by HIV-1 genome. It is responsible for two                 slow as ~0.05 sec-1. The slow release of hydrolysis products
biological activities that contribute to the pathogenicity of              from the protein ensures that the cycling of this machine will
HIV-1 infections in humans, and these activities are associated            continue even at high ATP concentrations. We suggest that
with separate domains of the protein. The cytoplasmic                      slow product release may be a general property of molecular
domain in the C-terminal modulates CD4 degradation via the                 machines, setting them apart from regular enzymes. Namely,
phosphorylation of two serine residues (S52 and S56). The                  while enzymes are turnover-optimized, molecular machines
N-terminal transmembrane domain forms oligomer, displays                   are cycling-optimized.
ion-channel activity, and is responsible for enhancing the
release of the virus particle. In order to determine the structure         296
of Vpu in lipid environments, a combination of solution-state
and solid-state NMR techniques is being applied to several                 Discrimination of the Structure of the Amino Acid by the
different constructs, including the trans-membrane domain (2-              Translational Machinery
37), cytoplasmic domain (28-81) and Vpu (2-51) in isotropic                Amanda Olivo, Josh Avins, Philip Effraim, Michael
micelles and aligned bilayer environments. Progress towards                Englander, Rachel Fleisher, Jiangning Wang, Ruben L.
the structure determination of Vpu will be presented.                      Gonzalez, Virginia Cornish
                                                                           Chemistry, Columbia University, 3000 Broadway, New York,
                                                                           NY, 10027
Molecular Machines: Function and                                           The involvement of tRNA structural elements beyond the
Assembly (295 – 300)                                                       anticodon in aminoacyl-tRNA selection by the ribosome
                                                                           has revealed that substrate recognition is considerably
295                                                                        more complex than originally envisioned in the adaptor
                                                                           hypothesis. By combining recent breakthroughs in
Out-of-equilibrium conformational cycling of GroEL                         aminoacyl-tRNA synthesis and mechanistic and structural
under saturating ATP concentrations                                        studies of protein synthesis, we are investigating whether
Gabriel Frank1, Gilad Haran2, Amnon Horovitz1                              aminoacyl-tRNA recognition further extends to the amino
  Structural Biology, 2Chemical Physics, Weizmann Institute                acid, which would explain various translation disorders
of Science, P.O.Box 26, Rehovot, 76100, Israel                             exhibited by misacylated tRNAs. Recently, we found that
The GroE chaperone system of E. coli is comprised of GroEL                 natural amino acids misacylated onto natural but non-native
and GroES. GroEL consists of 14 identical ~57 kDa subunits                 tRNAs are selected with efficiencies very similar to those of
arranged in two heptameric rings. It is a molecular machine                their correctly acylated counterparts. Despite this, small but
that facilitates protein folding. During the folding cycle,                reproducible differences in selection indeed demonstrate that
the system undergoes dramatic conformational changes                       the translational machinery is sensitive to the amino acid-
associated with the binding, hydrolysis and release of the                 tRNA pairing.
nucleotides.                                                               Here we present further progress defining the amino
GroEL exists in at least two allosteric states, T and R, which             acid specificity of the translational machinery, testing
interconvert in an ATP-controlled manner. Thermodynamic                    our hypothesis that discrimination becomes increasingly
analysis suggests that the T-state population becomes                      significant as the amino acid is further altered in structure.
negligible with increasing ATP concentrations, in conflict
with the requirement for conformational cycling, which is                  297
essential for the operation of molecular machines. To solve
this conundrum, we performed fluorescence correlation                      The cytosolic domain of mitochondrial fission protein,
spectroscopy on the single-ring version of GroEL, using a                  Fis1, reversibly binds and inserts into lipid membranes to
fluorescent switch recently built into its structure, which turns          affect membrane morphology
‘on’, i.e. increases its fluorescence dramatically, when ATP is            Robert Wells1, Lora Picton1, Marijke Koppenol1, Ananya
added. A series of correlation functions was measured as a                 Mujumdar2, R Blake Hill1
function of ATP concentration and analyzed using singular-
                                                                            Biology, 2Chemistry, Johns Hopkins University, 3400 North
                                                                           Charles Street, Baltimore, MD, 21211


Mitochondrial and peroxisomal fission are regulated by                    like shell assembled from several thousand copies of
a conserved set of proteins that mediate a high degree of                 bacterial microcompartment (BMC) subunit homologues.
membrane curvature to promote scission of the membrane.                   The shells enclose particular, sequentially acting enzymes
Despite the importance to cellular function and human health,             from various metabolic pathways; currently seven distinct
these processes are not well understood at the molecular                  metabolic categories have been identified. Here, we report
level. We are addressing this problem by using biophysical                on the crystal structures of multiple shell proteins from a
and biochemical experiments to determine the molecular                    propanediol utilizing (Pdu) microcompartment in Salmonella
mechanism of mitochondrial and peroxisomal fission. These                 enterica Typhimurium. The Pdu microcompartment
processes are mediated by at least two proteins: Drp1, a                  incorporates six paralogous shell subunits that, according to
mechanoenzyme that belongs to the dynamin superfamily                     their crystal structures, fulfill a rather sophisticated range of
and Fis1, a mitochondrial protein thought to recruit Drp1                 organizational and substrate transport functions. The findings
to sites of scission. We are testing the hypothesis that Fis1             illuminate the structure of a likely pore for transporting the
can stabilize membrane deformations important in fission,                 three-carbon substrates and products across the shell protein
using a membrane dependent conformational change that                     barrier. Structural data suggest that another Pdu shell protein
is modulated by the membrane surface charge. To do this                   is redox sensitive, and perhaps shuttles electrons across the
we are measuring Fis1 membrane binding and evaluating                     shell in order to regenerate encapsulated redox cofactors. The
changes in protein and membrane structure in different                    emerging structures of this remarkable class of subcellular
membrane environments. We found that the Fis1 cytosolic                   machines will be surveyed with an emphasis on new findings
domain alone is able to bind vesicles under physiological                 and principles.
membrane conditions. This binding is enhanced at lower                    Funding for this project provided by the NIH grant
pH and in the presence of negatively charged lipid head                   AI081146
groups. Our fluorescence and circular dichroism data
suggests a model in which the cytosolic domain undergoes                  299
a pH-dependent conformational change. Preliminary NMR
data has identified residues important in the pH-dependent                Discrimination of the Structure of the Amino Acid by the
membrane insertion. Our light scattering data, along with                 Translational Machinery
light and electron microscopy, show Fis1 is able to promote               Rachel Fleisher, Joshua Avins, Philip Effraim, Michael
membrane aggregation and deformation. Together, these                     Englander, Amanda Olivo, Jiangning Wang, Ruben Gonzalez,
data suggest that Fis1 can modulate membrane morphology                   Virginia Cornish
through a conformation change that may promote membrane                   Department of Chemistry, Columbia University, 3000
fission. Intriguingly, our results with Fis1 are similar to other         Broadway, New York, NY, 10027
tail-anchored mitochondrial proteins suggesting that our                  The involvement of tRNA structural elements beyond the
studies with fission proteins may reveal general principles               anticodon in aminoacyl-tRNA selection by the ribosome
of protein-membrane interactions that are important for                   has revealed that substrate recognition is considerably
forming and maintain membrane architecture. This work                     more complex than originally envisioned in the adaptor
was gratefully supported by NIH-RO1GM067180.                              hypothesis. By combining recent breakthroughs in
                                                                          aminoacyl-tRNA synthesis and mechanistic and structural
298                                                                       studies of protein synthesis, we are investigating whether
Mechanisms and atomic structures of the shell proteins                    aminoacyl-tRNA recognition further extends to the amino
from a bacterial organelle in Salmonella enterica                         acid, which would explain various translation disorders
Christopher Crowley1, Jeff Kopstein2, Fang Sheng3, Michael                exhibited by misacylated tRNAs. Recently, we found that
Sawaya3, Duilio Cascio3, Tom Bobik4, Todd Yeates2,3                       natural amino acids misacylated onto natural but non-native
  Molecular Biology Interdepartmental Program, 2Department                tRNAs are selected with efficiencies very similar to those of
of Chemistry and Biochemistry, 3Department of Energy                      their correctly acylated counterparts. Despite this, small but
Institute for Genomics and Proteomics, UCLA, 611 Charles                  reproducible differences in selection indeed demonstrate that
Young Dr. E., Los Angeles, CA, 90095, 4Biochemistry,                      the translational machinery is sensitive to the amino acid-
Biophysics and Molecular Biology, Iowa State University,                  tRNA pairing. Here we present further progress defining the
1210 Molecular Biology Building, Ames, IA, 50011                          amino acid specificity of the translational machinery, testing
                                                                          our hypothesis that discrimination becomes increasingly
In many bacteria, the enzymes in certain reaction pathways                significant as the aminoacyl-tRNA proceeds past aminoacyl-
form very large intracellular assemblies that serve as simple             tRNA selection to subsequent steps in the elongation cycle
metabolic organelles, generally referred to as “bacterial                 of translation.
microcompartments”. Instead of membrane-enclosed bodies,
their general structures are reminiscent of icosahedral or
spherical virus particles, comprised of an outer capsid-


300                                                                     and following purification of full-length CaM or individual
                                                                        domains, individual cysteines were: i) used to specifically
The ribosome discriminates the structure of the amino                   immobilize the N- or C-domains of CaM onto solid
acid at its peptidyltransferase center                                  supports or ii) covalently labeled with fluorescent probes
Joshua Avins, Michael Englander, Ruben Gonzalez, Virginia               to respectively permit binding measurements using either
Cornish                                                                 surface plasmon responance (SPR) or fluorescence correlation
Columbia University, 3000 Broadway, New York, NY,                       (FCS) spectroscopy. Equilibrium binding and kinetic rates
10027                                                                   were measured between CaM and target peptides engineered
The involvement of tRNA structural elements beyond                      onto the maltose binding protein scaffold. Complementary
the anticodon in aminoacyl-tRNA (aa-tRNA) selection                     molecular dynamics simulations permitted a mechanistic
by the ribosome has revealed that substrate recognition is              understanding of quantitative differences in binding energetics.
considerably more complex than originally envisioned in                 The rapid formation of an intermediate state is interpreted in
the adaptor hypothesis. We tested the possibility that this             terms of a model involving formation of an intermediate state
recognition extends all the way to the amino acid itself                that undergoes a subsequent conformational rearrangement
by synthesizing misacylated tRNAs altered only in the                   between the bound CaM and peptide to form the productive
stereochemistry of the amino acid. Using a defined in vitro             complex. Substantial differences in both the binding kinetics
translation system we have found that D-aa-tRNAs induce                 and equilibrium affinity constants are observed depending
significant translation disorders at the peptidyltransferase            on the peptide binding sequence, where enhanced binding
center that cause abortive translation in a subpopulation               correlates with the helix propensity of the peptide and the
of ribosomes the fraction of which depends on the identity              proximity between the N- and C-domains of CaM following
of the amino acid side chain. This amino acid-dependent                 peptide binding that together act to increase the on-rate of
perturbation of reactivity of the peptidyltransferase center is         CaM and decrease the entropic penalty of complexation.
consistent with increasing evidence suggesting that structural
rearrangements of the peptidyltransferase center gate the               302
chemistry of peptide bond formation. Furthermore, these
results provide a compelling mechanistic rationale not only             Identification of molecular determinants of secretin for
for the widespread observation that unnatural amino acids               binding and activation of its receptor
reduce the yield of protein synthesis but also for the highly           Maoqing Dong, Angela Le, Delia Pinon, Andrew Bordner,
conserved requirement for D-aa-tRNA deacylase enzymes.                  Laurence Miller
                                                                        Mayo Clinic, 13400 E. Shea Blvd., Scottsdale, AZ, 85259
                                                                        Secretin, whose receptor is a prototypic member of family B
Peptide Biochemistry and Protein-                                       G protein-coupled receptors containing many important drug
Peptide Interactions (301 – 308)                                        targets, is a linear 27-residue peptide hormone that controls
                                                                        pancreatic and biliary bicarbonate and water secretion.
                                                                        Although its carboxyl-terminal region is most important for
                                                                        high affinity binding and its amino-terminal region is critical
Molecular Recognition Between Individual Domains of                     for receptor selectivity and activation, it appears that binding
Calmodulin and Target Binding Sequences Involves a                      determinants are distributed throughout the entire length of
Kinetically Distinct Intermediate State                                 secretin. In this work, we have systematically investigated the
Curt Boschek1, Yijia Xiong1, Dayle M Smith2, Cheryl Baird1,             role of each position of this peptide in binding and activation
Thomas Squier1                                                          of its receptor using alanine scanning. Crucial residues for
  Cell Biology & Biochemistry, 2Computational Biol &                    binding affinity were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10,
Bioinfor, Pacific Northwest National Laboratory, 902                    Asp15, Leu19, and Leu23, with Ser2, Thr5, Glu9, Arg12, Leu13,
Battelle Boulevard, P.O. Box 999, Richland, WA 99352 USA,               Gln14, Arg18, Leu22, Leu26 and Val27 also contributing. The
Richland, WA, 99352                                                     most important residues for biological activity included His1,
To understand the mechanisms responsible for the high-                  Asp3, Gly4, Phe6, Thr7, Ser8, Glu9, Leu10, Asp15, Leu19, Leu22,
affinity interaction between the calcium signaling protein              and Leu23. Some of these residues (His1, Asp3, Phe6, Thr7,
calmodulin (CaM) and highly variable CaM-binding                        Asp15, and Leu23) are conserved throughout the family, while
sequences present in over 300 different target proteins, we             others are secretin-specific (Gly4 and Leu19). In an attempt to
have investigated the binding kinetics between CaM and                  define the active conformation of the highly flexible secretin
three different peptides corresponding to the CaM-binding               peptide, molecular modeling was used to predict where a
sequences of skeletal myosin light chain kinase (skMLCK),               disulfide bond could be introduced into its amino terminus
the Ca-ATPase (C28W), and the ryanodine receptor (RyR).                 to establish a backbone structure similar to that found in
Mutations at either T34C or T110C in CaM were constructed,              the NMR structure of receptor-bound pituitary adenylate
                                                                        cyclase-activating polypeptide. Based on this, we made a


series of secretin analogues incorporating cysteine pairs in             304
positions 2-7, 3-5, 3-6, 4-7, 7-9, and 4-10. Only the analogue
with a disulfide bond between positions 7 and 9 had affinity             The Proenkephalin Prohormone Displays Intact Protein-
and potency similar to natural secretin. Taken together, these           Dependent Differential Accessibilities of Multiple Protease
findings provide a guide for designing selective agonist                 Cleavage Site Domains to the Aqueous Environment
and antagonist ligands that could also act as leads for the              Weiya Lu1, Tong Liu3, Sheng Li3, Virgil Woods3, Vivian
development of highly potent small-molecule drugs acting at              Hook1,2
the orthosteric site.
                                                                          School of Pharmacy, 2Depts. of Neuroscience and
                                                                         Pharmacology, University of California, San Diego, 9500
*This work was supported by a grant from the National                    Gilman Dr. MC 0744, La Jolla, CA, 92093-0744, 3School of
Institutes of Health (DK46577)                                           Medicine, University of California, San Diego, 9500 Gilman
                                                                         Dr. MC 0656, La Jolla, CA, 92093-0656
                                                                         The enkephalin opioid peptide neurotransmitter, as well as
De Novo Design of Antimicrobial Peptides with Decreased                  other neuropeptides, is generated by proteolytic processing of
Binding to Human Serum Proteins                                          their prohormone precursors. Multiple dibasic cleavage sites
Zhe Yan1, Shaun Bevers1, Ziqing Jiang1, Michael Vasil2,                  within proenkephalin (PE) undergo proteolytic processing to
Robert Hodges1                                                           produce enkephalin neuropeptides. These cleavage sites have
  Department of Biochemistry and Molecular Genetics,                     been demonstrated to undergo sequential processing. Little
University of Colorado, School of Medicine, 12801 E                      is known about their conformational features and relative
17th Ave, Bldg RC-1,Room S9403, Aurora, CO, 80010,                       accessibilities to the aqueous environment. Therefore, the
  Department of Microbiology, University of Colorado, School             goal of this study was to investigate the relative accessibilities
of Medicine, 12801 E 17th Ave, Aurora, CO, 80010                         of the dibasic cleavage sites of recombinant human PE by
The activity of antimicrobial peptides in the blood is                   hydrogen-deuterium (H-D) exchange mass spectrometry
influenced by peptide stability (resistance to degradation               (DXMS). An HPLC quenched-flow system was developed
by proteases) and binding to serum proteins. The extent                  to observe both the rapid H-D exchange of PE domains at
of binding to serum proteins can have a significant impact               subsecond time resolution and slower rates of exchange
on pharmacokinetic parameters, such as clearance rates. In               of other subregions of PE. Significant results showed
addition, since only the unbound drug is available to interact           differential relative accessibilities of dibasic processing sites
with the therapeutic target, the extent of serum binding can             of proenkephalin (PE) to the aqueous solvent environment.
have significant effects on pharmacodynamic properties.                  The differences in relative accessibilities of the cleavage
Thus, the extent of antimicrobial peptide binding to serum               sites to the aqueous environment suggests differential tertiary
proteins must be controlled. In our study, antimicrobial                 conformational features of these cleavage site domains.
activity (minimal inhibitory concentration, MIC) of our lead             However, peptides derived from PE, by pepsin digestion,
peptide, D1, was dramatically less in the presence of serum              subjected to DXMS analyses overall show faster rates of
proteins. Thus, we purified and identified a key serum protein           H-D exchange. These results indicate the lower accessibility
(apolipoprotein A1, Apo A1) responsible for D1 binding                   of cleavage site domains of intact PE, compared to peptides
using affinity chromatography. Apo A1 was shown to bind                  derived from PE. The tertiary conformational features of
D1 specifically by isothermal titration calorimetry (ITC),               PE, thus, are apparently absent in the peptides derived from
surface plasmon resonance spectroscopy (SPR) and enzyme-                 PE (after pepsin digestion). These data demonstrate that the
linked immunosorbent assay (ELISA). We then designed                     tertiary conformation of the intact PE protein participates in
new antimicrobial peptides with reduced binding properties               the differential accessibilities of the multiple dibasic protease
to Apo A1 and other serum proteins. Peptide D16 was shown                cleavage sites of PE. The results presented here establish
to have the desired properties of low affinity binding to Apo            the differential conformational features of multiple cleavage
A1 and other serum proteins while maintaining excellent                  sites of intact PE, which require the tertiary structure of
antimicrobial activity against the gram-negative bacteria                proenkephalin.
(Pseudomonas aeruginosa, Pa, and Acinetobacter baumannii,
Ab), negligible toxicity (as measured by hemolytic activity to
human red blood cells) and outstanding therapeutic indices
of 3,355 against Ab and 895 against Pa. This study clearly
demonstrates that prior to carrying out animal efficacy studies,
antimicrobial peptides should be screened for the following
properties: excellent antimicrobial activity and low toxicity
to human cells resulting in high therapeutic indices as well as
weak binding affinity to serum proteins.


305                                                                     306
Semi-synthesis of glutamate receptor associated                         Targeting HIV-1 Envelope Proteins Utilizing a Multivalent
proteins – Structure and function relationship study of                 Approach
PDZ-domains                                                             Ping Wang1, Min Zhou1, Brian Wellensiek2, Nafees Ahmad2,
Søren Wittrup Pedersen, Anders Bach, Anders Skov                        Indraneel Ghosh1
Kristensen, Kristian Strømgaard                                         1
                                                                         Department of Chemistry and Biochemistry, University
Department of Medicinal Chemistry, University of                        of Arizona, 1306 E. University Blvd., Tucson, AZ, 85721,
Copenhagen, Jagtvej 162, Copenhagen, 2100, Denmark                      2
                                                                         Department of Immunobiology, University of Arizona, 1501
The ionotropic glutamate receptors (iGluR) are essential for            N. Campbell Ave., Tucson, AZ, 85724
basic neurotransmission and disturbances of this receptor               Envelope glycoprotein spikes on the surface of the HIV-1
system have devastating consequences leading to a number                retrovirus target host cells through a homotrimer of gp120/
of diseases in the brain. Recently, it has been realized that           gp41 heterodimers. The homotrimer engages the cellular
iGluRs are part of complex signalling networks, primarily by            receptors CD4 and CCR5/CXCR4 and mediates the fusion
interacting with intracellular proteins. These include the so-          of the viral and the target cellular membranes. F23[1], a
called PDZ domains which could be potential drug targets.               designed CD4 mimetic protein, based on the scorpion-toxin
The PDZ domains consist of approximately 100 amino acids                scaffold has been previously utilized for virus neutralization.
and are distributed throughout the proteome. X-ray studies              Based on the structure of trimeric gp120,[2-3] a large series
indicate that the domains are able to bind intracellular                of multivalent F23 inhibitors were designed and tested for
C-terminals through a canonical interaction with 3 backbone             their ability to block the viral entry.. The best trivalent ligand
amides located at a conserved GLGF-site. But the importance             showed ~60 % viral entry inhibition at 200 pM with >1000-
of the individual amides is still unknown.                              fold improvement over the monovalent F23, making it one
By applying expressed protein ligation it is possible to semi-          of the most potent entry inhibitors identified to date. We will
synthesize the PDZ2 domain of PSD-95 scaffolding protein                also discuss the surprising length requirements for the F23-
thereby allowing incorporation of amide-to-ester mutation.              based multivalent ligands as well as new strategies for viral
This modification removes the hydrogen donor ability at                 inhibition utilizing heterobivalent approaches, where two
one of the three amide sites in the GLGF-site thus making               distinct regions on the viral surface are targeted.
it possible to discriminate between their individual binding            This research was supported by R01AI068414 (NIH)
contributions in a functional assay.                                    Reference:
Four thioester peptides, resembling the N-terminal of PSD95             1. Huang, C. C.; Stricher, F.; Martin, L.; Decker, J. M.;
PDZ2, were synthesized using automated BOC solid phase                     Majeed, S.; Barthe, P.; Hendrickson, W. A.; Robinson,
peptide synthesis. One peptide had the WT sequence and                     J.; Roumestand, C.; Sodroski, J.; Wyatt, R.; Shaw, G. M.;
functioned as a control, while three were synthe