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									Protein Methods II

               Andy Howard
         Introductory Biochemistry
               Fall 2009, IIT
 Proteins are worth studying
   We’ll finish our quick overview of
    methods of studying proteins




09/10/09      Biochemistry: Protein Methods II   p. 2 of 36
 Plans
   Purification methods
   Analytical methods
   Structural methods




09/10/09      Biochemistry: Protein Methods II   p. 3 of 36
       Ion-exchange
       chromatography
  Charged species affixed to
   column
  Phosphonates (-) retard
   (+)charged proteins:
   Cation exchange
  Quaternary ammonium salts
   (+) retard (-)charged
   proteins: Anion exchange
  Separations facilitated by
   adjusting pH
09/10/09     Biochemistry: Protein Methods II   p. 4 of 36
   Affinity chromatography
          Stationary phase contains a species that
           has specific favorable interaction with
           the protein we want
          DNA-binding protein specific to
           AGCATGCT: bind AGCATGCT to a
           column, and the protein we want will
           stick; every other protein falls through
          Often used to purify antibodies by
           binding the antigen to the column

09/10/09            Biochemistry: Protein Methods II   p. 5 of 36
           Metal-ion affinity
           chromatography
 Immobilize a metal ion, e.g. Ni, to
  the column material
 Proteins with affinity to that metal
  will stick
 Wash them off afterward with a
  ligand with even higher affinity
 We can engineer proteins to
  contain the affinity tag:
  poly-histidine at N- or C-terminus

09/10/09         Biochemistry: Protein Methods II   p. 6 of 36
 High-performance liquid
 chromatography
 Many LC separations can happen faster
  and more effectively under high
  pressure
 Works for small molecules
 Protein application is routine too, both
  for analysis and purification
 FPLC is a trademark, but it’s used
  generically

09/10/09    Biochemistry: Protein Methods II   p. 7 of 36
 Electrophoresis

            Separating analytes by charge
             by subjecting a mixture to a
             strong electric field
            Gel electrophoresis: field
             applied to a semisolid matrix
            Can be used for charge
             (directly) or size (indirectly)

09/10/09           Biochemistry: Protein Methods II   p. 8 of 36
           SDS-PAGE
              Sodium dodecyl sulfate: strong detergent,
               applied to protein
              Charged species binds quantitatively
              Denatures protein
               – Good because initial shape irrelevant
               – Bad because it’s no longer folded
              Larger proteins move slower because they
               get tangled in the matrix
              1/Velocity  √MW



09/10/09                 Biochemistry: Protein Methods II   p. 9 of 36
           SDS PAGE illustrated




09/10/09         Biochemistry: Protein Methods II   p. 10 of 36
           Isoelectric focusing I

 Protein applied to gel without
  charged denaturant
 Electric field set up over a
  pH gradient (typically pH 2 to
  12)
 Protein will travel until it
  reaches the pH where
  charge =0 (isoelectric point)
09/10/09         Biochemistry: Protein Methods II   p. 11 of 36
 Isoelectric focusing II
 Sensitive to single changes in
  charge (e.g. asp -> asn)
 Can be readily used preparatively
  with samples that are already
  semi-pure




09/10/09    Biochemistry: Protein Methods II   p. 12 of 36
           Ultraviolet spectroscopy
          Tyr, trp absorb and fluoresce:
           abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr)
          Reliable enough to use for estimating protein
           concentration via Beer’s law
          UV absorption peaks for cofactors in various
           states are well-understood
          More relevant for identification of moieties
           than for structure determination
          Quenching of fluorescence sometimes
           provides structural information

09/10/09             Biochemistry: Protein Methods II   p. 13 of 36
 Warning: Specialty Content!

 I determine protein structures (and develop
  methods for determining protein structures)
  as my own research focus
 So it’s hard for me to avoid putting a lot of
  emphasis on this material
 But today I’m allowed to do that, because it’s
  one of the stated topics of the day.



09/10/09       Biochemistry: Protein Methods II   p. 14 of 36
 How do we determine structure?
 We can distinguish between methods
  that require little prior knowledge
  (crystallography, NMR, ?CryoEM?)
  and methods that answer specific
  questions (XAFS, fiber, …)
 This distinction isn’t entirely clear-cut




09/10/09      Biochemistry: Protein Methods II   p. 15 of 36
 Crystallography: overview
 Crystals are translationally ordered 3-D
  arrays of molecules
 Conventional solids are usually crystals
 Proteins have to be coerced into
  crystallizing
 … but once they’re crystals, they
  behave like other crystals, mostly

09/10/09     Biochemistry: Protein Methods II   p. 16 of 36
 How are protein crystals
 unusual?
 Aqueous interactions required for
  crystal integrity: they disintegrate if dried
 Bigger unit cells (~10nm, not 1nm)
 Small # of unit cells and static disorder
  means they don’t scatter terribly well
 So using them to determine 3D
  structures is feasible but difficult


09/10/09      Biochemistry: Protein Methods II   p. 17 of 36
 Crystal structures: Fourier
 transforms of diffraction results
          Experiment:
           – Grow crystal, expose it to X-ray
           – Record diffraction spots
           – Rotate through small angle and repeat ~180 times
          Position of spots tells you size, shape of unit
           cell
          Intensity tells you what the contents are
          We’re using electromagnetic radiation, which
           behaves like a wave, exp(2ik•x)
          Therefore intensity Ihkl = C*|Fhkl|2

09/10/09              Biochemistry: Protein Methods II   p. 18 of 36
 What are these Fhkl values?
   Fhkl is a complex coefficient in the Fourier
    transform of the electron density in the unit
    cell:
    (r) = (1/V) hkl Fhkl exp(-2ih•r)
   Critical point: any single diffraction spot
    contains information derived from all the
    atoms in the structure; and any atom
    contributes to all the diffraction spots


09/10/09        Biochemistry: Protein Methods II   p. 19 of 36
    The phase problem
                                                       Fhkl
 Note that we said Ihkl = C*|Fhkl                |2             bhkl
                                                         
 That means we can figure out                           ahkl
    |Fhkl| = (1/C)√Ihkl
   We can’t figure out the direction of F:
  Fhkl = ahkl + ibhkl = |Fhkl|exp(ihkl)
 This direction angle is called a phase angle
 Because we can’t get it from Ihkl, we have a
  problem: it’s the phase problem!

09/10/09            Biochemistry: Protein Methods II     p. 20 of 36
           What can we learn?
   Electron density map + sequence  we can
    determine the positions of all the non-H
    atoms in the protein—maybe!
   Best resolution possible: Dmin =  / 2
   Often the crystal doesn’t diffract that well, so
    Dmin is larger—1.5Å, 2.5Å, worse
   Dmin ~ 2.5Å tells us where backbone and
    most side-chain atoms are
   Dmin ~ 1.2Å: all protein non-H atoms, most
    solvent, some disordered atoms; some H’s

09/10/09          Biochemistry: Protein Methods II   p. 21 of 36
           What does this look like?

     Takes some
      experience to
      interpret
     Automated
      fitting
      programs work
      pretty well with         ATP binding to a protein of
      Dmin < 2.1Å              unknown function: S.H.Kim

09/10/09          Biochemistry: Protein Methods II   p. 22 of 36
           How’s the field changing?
    1990: all structures done by professionals
    Now: many biochemists and molecular
     biologists are launching their own
     structure projects as part of broader
     functional studies
    Fearless prediction: by 2020:
      – crystallographers will be either technicians or
        methods developers
      – Most structures will be determined by cell
        biologists & molecular biologists
09/10/09          Biochemistry: Protein Methods II   p. 23 of 36
           Macromolecular NMR
          NMR is a mature field
          Depends on resonant interaction between EM
           fields and unpaired nucleons (1H, 15N, 31S)
          Raw data yield interatomic distances
          Conventional spectra of proteins are too
           muddy to interpret
          Multi-dimensional (2-4D) techniques:
           initial resonances coupled with additional ones


09/10/09              Biochemistry: Protein Methods II   p. 24 of 36
     Typical protein 2-D spectrum

      Challenge:
       identify which
       H-H distance is
       responsible for a
       particular peak
      Enormous
       amount of
       hypothesis                  Prof. Mark Searle,
       testing required            University of Nottingham
09/10/09        Biochemistry: Protein Methods II    p. 25 of 36
           Results of NMR studies
   Often there’s a family of structures that
    satisfy the NMR data equally well
   Can be portrayed as a series of threads
    tied down at unambiguous assignments
   They portray the protein’s structure in
    solution
   The ambiguities partly represent real
    molecular diversity; but they also represent
    atoms that area in truth well-defined, but
    the NMR data don’t provide the
    unambiguous assignment
09/10/09         Biochemistry: Protein Methods II   p. 26 of 36
     Comparing NMR to X-ray
   NMR family of structures often reflects real
    conformational heterogeneity
   Nonetheless, it’s hard to visualize what’s
    happening at the active site at any instant
   Hydrogens sometimes well-located in NMR;
    they’re often the least defined atoms in an X-
    ray structure
   The NMR structure is obtained in solution!
   Hard to make NMR work if MW > 35 kDa

09/10/09          Biochemistry: Protein Methods II   p. 27 of 36
    What does it mean when NMR
    and X-ray structures differ?
    Lattice forces may have tied down or moved
     surface amino acids in X-ray structure
    NMR may have errors in it
    X-ray may have errors in it (measurable)
    X-ray structure often closer to true atomic
     resolution
    X-ray structure has built-in reliability checks


09/10/09         Biochemistry: Protein Methods II   p. 28 of 36
     Cryoelectron
     microscopy
  Like X-ray crystallography,
   EM damages the samples
  Samples analyzed < 100K
   survive better
  2-D arrays of molecules
       – Spatial averaging to improve
         resolution
       – Discerning details ~ 4Å resolution
     Can be used with crystallography

09/10/09           Biochemistry: Protein Methods II   p. 29 of 36
           Circular dichroism
 Proteins in solution can
  rotate polarized light
 Amount of rotation varies
  with 
 Effect depends on
  interaction with secondary
  structure elements, esp. 
 Presence of characteristic
   patterns in presence of
  other stuff enables
  estimate of helical content
09/10/09        Biochemistry: Protein Methods II   p. 30 of 36
    Poll question:
    discuss!                                     QuickTime™ an d a
                                                                                Sperm
                                        TIFF (Uncompressed) decompressor
                                           are need ed to see this p icture .
                                                                                whale
   Which protein would yield                                                   myoglobin
    a more interpretable CD                                                     PDB 2jho
    spectrum?                                                                   1.4Å
                                                                                16.9 kDa
    – (a) myoglobin
    – (b) Fab fragment of
      immunoglobulin G                       QuickTime™ an d a
                                                                      Anti-
    – (c) both would be fully       TIFF (Uncompressed) decompressor  fluorescein
                                       are need ed to see this p icture .
                                                                      Fab
      interpretable
                                                                      PDB 1flr
    – (d) CD wouldn’t tell us                                         1.85 Å
      anything about either                                           52 KDa
      protein
09/10/09          Biochemistry: Protein Methods II                     p. 31 of 36
           Ultraviolet spectroscopy
   Tyr, trp absorb and fluoresce:
    abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr)
   Reliable enough to use for estimating protein
    concentration via Beer’s law
   UV absorption peaks for cofactors in various
    states are well-understood
   More relevant for identification of moieties than
    for structure determination
   Quenching of fluorescence sometimes provides
    structural information

09/10/09         Biochemistry: Protein Methods II   p. 32 of 36
       X-ray spectroscopy

 All atoms absorb UV or
  X-rays at characteristic
  wavelengths
 Higher Z means higher
  energy, lower  for a
  particular edge


 09/10/09     Biochemistry: Protein Methods II   p. 33 of 36
 X-ray spectroscopy II

 Perturbation of absorption
  spectra at E = Epeak +  yields
  neighbor information
 Changes just below the peak
  yield oxidation-state
  information
 X-ray relevant for metals, Se, I


09/10/09      Biochemistry: Protein Methods II   p. 34 of 36
           Solution scattering
   Proteins in solution scatter X-rays in
    characteristic, spherically-averaged ways
   Low-resolution structural information
    available
   Does not require crystals
   Until ~ 2000: needed high [protein]
   Thanks to BioCAT, SAXS on dilute
    proteins is becoming more feasible
   Hypothesis-based analysis



09/10/09           Biochemistry: Protein Methods II   p. 35 of 36
           Fiber
           Diffraction

  Some proteins, like many
   DNA molecules, possess
   approximate fibrous order
   (2-D ordering)
  Produce characteristic fiber
   diffraction patterns
  Collagen, muscle proteins,
   filamentous viruses

09/10/09          Biochemistry: Protein Methods II   p. 36 of 36

								
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