Experimental mapping of protein precipitation diagrams

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					            Experimental mapping of
          protein precipitation diagrams
Morten O.A. Sommer (morten@ccs.ki.ku.dk)
Centre for Crystallographic Studies
University Of Copenhagen
                                    Look at protein crystallography
                                         and liquid handling
                              Liquid handling for protein crystallization            Low volume liquid
                                                                                     handling
                             1000
                                                                                     technology 
experiments pr micro liter




                                                                                     more experiments
  Number of chemical




                              100
                                                                                     performed using
                                                                                     less SAMPLE
                               10
                                                                                     Lab automation
                                                                                      more
                                1                                                    experiments
                                    1994



                                           1996



                                                  1998



                                                         2000



                                                                2002



                                                                       2004



                                                                              2006
                                                                                     performed using
                                                                                     less TIME
           Low TIME and SAMPLE consumption enables new
           approaches to protein crystallization
                    Microfluidic formulator
                         technology
                                                      1 mm




Experiments done by: Carl Hansen, Morten Sommer and
Stephen Quake. PNAS (2004) 101:14431-14436
                                 Metering accurate and robust
                       4.5
                                                                       Metering accuracy
                        4                                              determined by absorption
                                                                       measurements
                       3.5
Injected volume [nL]




                                                                                   Motor oil
                        3
                                                      Water at     Raw Linseed oil (SAE 20) at
                       2.5                            20 degrees C 20 degrees C    20 degrees C

                        2

                       1.5

                        1
                                                                           Injection volume:
                       0.5                                                 80 pL +/- 0.6 pL
                        0
                             0     10     20     30      40      50

                                  Number of injection cycles
                                                Experiments done by: Carl Hansen, Morten Sommer and
                                                Stephen Quake. PNAS (2004) 101:14431-14436
         Ideal approach to protein
               crystallization
• GOAL: Further rationalization of protein
  crystallization
• Using minute amounts of protein sample
  to quantify:
  – Protein stability, folding & activity
  – Protein physical chemistry (solubility and
    precipitation limits)
  – Protein - protein interactions (Virial
    coefficients etc.)
                   Why use precipitation diagrams?


Phase diagram
of: aspartyl-
tRNA
synthetase-1
From Thermus
thermophilus
Zhu et. al. 2001
Acta Cryst. D
57:552-558
Detecting precipitation


                            Detection of Precipitation
                    25.00

                    20.00




            STDEV
                    15.00

                    10.00

                     5.00

                     0.00
                            0    10   20   30   40   50   60

                                 Titration Number
      Towards a rational approach:
      Tailor made screens based on
          precipitation diagrams
• Characterize protein
  solution and identify
  potential conditions


• Map protein precipitation
  diagrams

• Design and set up a tailor
  made crystallization
  screen based on the
  precipitation diagrams of
  the particular protein
         Initial validation: Xylanase
1. Make solubility
   fingerprint identifying
   precipitating
   chemical conditions
2. Map precipitation                          40


   diagrams for                               35


   potential conditions    Xylanase [mg/ml]
                                              30


                                              25
3. Set up crystallization                     20
   experiments near                           15

   precipitation                              10

   boundary                                    5


                             0
                                          Carl Hansen, Morten Sommer2 and
                   Experiments 0done by: 0.5         1       1.5
                                               Na/K 101:14431-14436
                   Stephen Quake. PNAS (2004) Tartrate [M]
Initial validation: Xylanase
 Crystallization probability pr. trial
 OPT (Tailor made screen): 27 hits out of 48
 experiments = 56 %
 Sparse matrix screens: 3 hits out of 384
 experiments = 0.8 %




              Experiments done by: Carl Hansen, Morten Sommer and
              Stephen Quake. PNAS (2004) 101:14431-14436
         Further validation
       Membrane protein: SERCA
• Study the
  crystallization of
  membrane proteins
  using the previously
  crystallized calcium
  pump (SERCA)
• Crystallization
  conditions are know
• Reliable preparation
  and purification
                         Sørensen et.al., (2004) Science 304, 1672-1675
            Further validation
         Membrane protein: SERCA
                                                   1



                                                  0.8
              Relative Precipitant Strength
                                                  0.6



                                                  0.4



                                                  0.2

• Solubility fingerprint can be used to identify specific
  protein – precipitant interactions
                     0
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• Identification of specific interaction between sodium


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                                                       as
  acetate and SERCA
                                                       m
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• Sodium acetate is an established crystallization agent for
  SERCA
                                                        Experiments done by: Morten Sommer and Sine Larsen.
                                                        Journ. of Synchrotron Rad. (2005) in press
           Further validation
        Membrane protein: SERCA
• Based on the characterization of specific protein
  – precipitant interactions several chemical
  conditions were selected for precipitation
  diagram mapping
• Set up tailor made crystallization screen
• Identification of well known and new
  crystallization agents
• Potentially useful for crystallizing previously
  uncrystallized membrane proteins

                  Experiments done by: Morten Sommer and Sine Larsen.
                  Journ. of Synchrotron Rad. (2005) in press
           Process diagram
                                       Analysis of
           Solubility              protein-precipitant
           fingerprint                 interaction



Protein
sample                                                      Precipitation
                                                             diagrams
                          Formulator
                             chip

                                                         Design rational
Crystals                                                  crystallization
                                                           experiments

                                           Setup
              Monitor
                                       crystallization
            experiments
                                        experiments
                     Perspectives
       Rational approach to protein crystallization
       using minute sample volumes

Task                                Volume consumption (μL)
Solubility characterization               10
Setup of 300 crystallization exp.         25

TOTAL                                     35
        Rational approaches are possible for
        many targets that are available in low
        amounts (Membrane proteins, protein
        complexes, and proteins purified from
        native tissue).
  Testing previously uncrystallized
        membrane proteins
The ultimate test of the rational approach:
3 previously uncrystallized membrane
proteins are tested.
   1. Voltage gated channel
   2. DsbB: disulfide bond forming
   membrane protein.
   3. AIDA: adhesin autotransporter
   protein
                                Voltage-gated channel:
                                  Solubility mapping
                                Voltage-gated channel
                            in 0.1 M Linear Buffer pH 6.5           Based on the
                   3
                                                                    solubility fingerprint
                                                                    40 precipitation
                  2.5
                                                                    diagrams are mapped
Protein [mg/ml]




                   2
                                                                    out.
                  1.5
                                                                    Volume consumption pr.
                   1                                                precipitation diagram:
                                                                    100 nL
                  0.5
                                                                    Total consumption for
                   0
                                                                    solubility screen and
                        0         5         10          15     20
                                                                    precipitation diagrams:
                                      PEG 400 [% w/v]
                                                                    8 μL (44.8 μg)
                                      Experiments done by: Morten Sommer, Jens-Christian Navarro
                                      Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
                             Voltage-gated channel:
                            Crystallization experiments
                                Voltage-gated channel
                                                                    A tailor made screen
                            in 0.1 M Linear Buffer pH 6.5           of 288 conditions is
                   3
                                                                    designed.
                                                                    The screen is set up
                  2.5
                                                                    as sitting drop exp.
Protein [mg/ml]




                   2
                                                                    using an ORYX 6 at
                  1.5                                               Douglas Instruments
                   1                                                using 17 μL sample
                  0.5
                                                                    (95 μg of protein)
                   0                                                An additional screen
                        0         5         10          15     20
                                                                    is set up testing
                                      PEG 400 [% w/v]
                                                                    different additives
                                      Experiments done by: Morten Sommer, Jens-Christian Navarro
                                      Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
               Voltage-gated channel:
              Crystallization experiments




                                               Crystals tested at
Scalebars =                                    ESRF beamline ID 29.
100 microns
                                                   Not protein crystals
                 Experiments done by: Morten Sommer, Jens-Christian Navarro
                 Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
                        DsbB:
                  Solubility mapping
40 chemical conditions                                         DsbB in 0.1 M Linear Buffer pH 9 and
                                                                     80 mM Calcium Acetate
are chosen for
determination of their                                 7

precipitation diagram.                                 6




                               Protein Conc. [mg/ml]
                                                       5
Using a total of 4 μL (40
μg of protein).                                        4

                                                       3

A tailor made screen                                   2

consisting of 288                                      1

conditions was                                         0
                                                           0           10          20          30     40
designed and set up                                                         PEG 4000 [% w/v]
using 18 uL (180 μg)
                 Experiments done by: Morten Sommer, Jens-Christian Navarro
                 Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
                            DsbB:
                 Crystallization experiments
          Crystals tested at
          ESRF ID 29
                                                          Scalebars =
  Some were                                               100 microns
  not protein.

Some did not
diffract  cryo
optimization




                      Experiments done by: Morten Sommer, Jens-Christian Navarro
                      Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
                                                     AIDA:
                                          Solubility characterization
                                                                          40 precipitation diagrams
                            AIDA with 0.1 M Linear Buffer pH 4 and
                                                                          are selected for mapping
                            AIDA with 0.1 M Linear Buffer pH 4 and
                                       8 % v/v Glycerol
                                       8 % v/v Glycerol                   based on solubility
                   5
                   5                                                      fingerprint
                  4.5
                  4.5
                   4
                   4
                  3.5                                                     Based on the diagrams a
Protein [mg/ml]




                  3.5
Protein [mg/ml]




                   3
                  2.5
                                                                          576 experiment screen is
                   2
                   2                                                      designed and set up
                  1.5
                  1.5
                   1
                  0.5
                  0.5                                                     Volume consumption:
                   0
                   0
                        0             5          10         15       20
                                                                          Solubility mapping: 8 μL
                        0             5          10         15       20
                                PEG 1500 monomethyl ether [% w/v]
                                PEG 1500 monomethyl ether [% w/v]



                                                                          Crystallization exp.: 22 μL
                                                  Experiments done by: Morten Sommer, Jens-Christian Navarro
                                                  Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
                        AIDA:
             Crystallization experiments
                     Some did not diffract
Crystals tested     optimize cryo conditions
at ESRF ID 29




                                                             Scalebars =
                                                             100 microns




                  Experiments done by: Morten Sommer, Jens-Christian Navarro
                  Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
                Protein consumption
                VGC            DsbB           AIDA
Solubility      >5000 exp.     >5000 exp.     >5000 exp.
char.           8 μL (45μg)    8 μL (80μg)    8 μL (80μg)
Cryst. Exp      ~576 exp.      ~ 288 exp.     ~ 576 exp.
                34 μL(190μg)   18 μL(180μg)   22 μL(220μg)
Crystal Hits    No             Yes            Yes

Total protein   235 μg         260 μg         300 μg
consumption
           Summarizing remarks
• As liquid handling technologies have achieved
  ~1 nL experimental volumes.
  A rationalization of protein crystallization in terms of
   precipitation diagrams is possible
• Rational approaches to protein crystallization are
  performed using < 300 μg of protein sample.
• Hope: This method and technology will allow for
  a better understanding of the crystallization
  process - and that complementary low volume
  technology will be developed to address other
  aspects of protein crystallization
                  Acknowledgements
• Univ. Of Copenhagen                • Stanford
   –   Jens-Christian Poulsen           – Prof. Stephen R. Quake
   –   Prof. Sine Larsen             • Univ. Of British Columbia
   –   Flemming Hansen                  – Ass. Prof. Carl L. Hansen
   –   Centre for Crystallographic   • Univ. of California – San Diego
       Studies
                                        – Prof. Mauricio Montal
• Univ. Of Aalborg                      – Dr. Jose Santos
   – Prof. Daniel Otzen              • Douglas Instruments
   – Brian Vad
                                        – James Smith
• Univ. Of Aarhus                       – Peter Baldock
   – Ass. Prof. Poul Nissen             – Patrick Shaw Stewart
   – Prof. Jesper Vuust Møller       • ESRF – ID29
• Tech. Univ. Of Denmark                – Gordon Leonard
   – Ass. Prof. Jörg Kutter
   – Detlef Snakenborg
         Experimental mapping of
       protein precipitation diagrams
Morten O.A. Sommer (morten@ccs.ki.ku.dk)
Centre for Crystallographic Studies
Univ. Of Copenhagen