Growing Protein Crystals

1
Growing Protein Crystals

• Using Calcium-Integrin Binding Protein (CIB) As A
  ModelPresented by
• Chad Blamey

FBP www.scripps.edu/arvai/ xtals/xtals.html
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Goals

• Understand how crystals grow
• Discus techniques for crystallizing proteins
• Strategies for optimizing crystal growth
• Obtaining crystals different conformations
• CIB as a model

3
CIB Facts

• CIB has two EF-hands
• Binds calcium
• CIB is a calcium induced protein regulator
• Like calmodulin
• 191 amino acids
• 21.7 kDa

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More CIB Facts

• No tryptophan
• Three cysteines
• No disulfide bonds
• Two surface exposed (Volgel et al., 2000)
• Recently found dimers and higher multimers form
• Formal charge 13, pI 5.3
• No known enzymatic activity

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CIB Physiology
Extra cellular matrix

• Binds ? cytoplasmic tail of integrin in the
  presence of Ca2
• Binds actin
• CIB regulates the connection between the extra
  cellular matrix and the cytoskeleton
• No evidence of an ?-integrin cytoplasmic
  tail-CIB-actin complex

Cytoskeleton
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Crystallization

• Crystals need to be of sufficient size and
  quality to diffract x-rays
• Size Normally should be 100 ?m in smallest
  dimension
• Quality Reflections collected from diffraction
  data are the primary source of data to build an
  electron density map, therefore quality of
  protein model depends greatly on crystal quality
• Growing good crystals is key to a good structure

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Crystallization

• With enzymes is is often important to maintain
  enzymatic activity in crystal
• Not necessary with CIB
• Best way to test crystal quality is by mounting a
  crystal and attempting to diffract x-rays
• Visual inspection helpful too

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Phases of Proteins In Solution
Not to be confused with phases of light
Crystals
Figure 3.3
Precipitation
Supersaturation
Growth Nucleation
Metastabile
Barrier of Nucleation
Undersaturated
Growth
Soluble protein
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Nucleation Growth
Basic concept

• Concentrate solution enough so nucleation occurs
  in only a few cases
• Initial growth pulls some protein out of solution
• Reducing protein back into metastable range
• Grow only a few large crystals

Phase diagram
Supersaturation
Metastabile
Undersaturated
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Growing Crystals Vapor Diffusion

• Hanging drop method
• Widely used
• Fast
• Simple
• Inexpensive
• ? Test for growth in a variety of conditions

Sitting drop
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Growing CrystalsOther Techniques
Ultracentrifugation

• Spin at extremely high speeds, hundreds of
  thousands of gs
• Slowly increases the relative protein
  concentration

Dialysis

• Uses liquid-liquid diffusion
• Diffusion is slow
• Rate controlled by membrane

12
Crystal Screens

• Hampton Research screen tests a wide assortment
  of conditions of salts, buffers, pHs and
  additives
• Best conditions from literature
• Often first hits with screens are small poor
  quality crystals
• Do not use the absence of crystals as a gauge of
  conditions rather use solubility

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Factors Effecting Crystal Growth
Most important

• Ionic Strength
• Specific Ions (Ca)
• Protein Concentration
• Detergents
• Inorganic Precipitant
• pH
• Temperature
• Time
• Monodispersion

• Vibrations
• Pressure
• Gravity
• Relative Proportion of Conditions
• Purity Of Protein
• Access to water
• Ligands
• Binding partners

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Optimize Crystal Growth

• The number of factors can be overwhelming
• Focus on those factors which most effect growth
• Set up arrays to vary two different conditions at
  once
• Cross your fingers

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The Tricky Part

• Conditions for crystallization are not
  independent of each-other
• Crystal quality will change as you vary growth
  conditions
• Figure 3.4

B
A
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Protein Conformations

• Apo CIB (EGTA treated)
• No hits, same as calmodulin
• Ca bound CIB
• Best luck so far
• Ca bound CIB ?IIb cytoplasmic tail
• Trials starting
• Ca bound CIB actin
• No trials yet

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CIB Protein Characterization
Further characterization of a protein can improve
crystal quality
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Ligands and Co-crystals

• One way to obtain crystals is with different
  ligands and/or co-crystallize with another
  protein, as with CIB
• Calcium, ? integrin peptide, actin
• Actin has been crystallized several times before
• Often changes protein conformation
• CIB bound to Ca2 is has a smaller radius than apo

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Small Crystals

• Often small crystals can be made larger by
  microseeding new drops with previously grown
  crystals or adding more protein solution
• Multinucleation can be avoided by reducing the
  temperature or adding glycerol
• Crystals only need to be large enough to diffract
  x-rays well

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Radical Approaches

• Remove either N- or C-terminus by weak
  proteolysis or by molecular cloning
• Often termini can be disordered which interferes
  with lattice formation
• Crystallize with a fusion protein
• Fusion proteins are well documented with a solved
  structure that easily from a lattice
• Pull the fusion protein into an ordered crystal
• Can use the protein for molecular replacement to
  solve phase
• Many recombinant proteins are purified using
  fusions anyways, i.e. not hard to try

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Radical Approaches, Cont

• Mutants
• if you know specific residues are giving you
  problems you can mutate the protein using
  recombinant DNA technology
• Radical approaches can result in conformations
  different from the native state
• Changing the conformation means starting over
  through the Hampton screens

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Discussion

• Given a hypothetical protein that doesnt give
  you any positive hits in your first Hampton
  screen what could you do to obtain quality
  crystals?
• Hint almost no precipitation in each drop!
• Say on the other hand at room temperature you
  have a condition with lots of tiny crystals, what
  can be done to reduce the amount of nucleation?

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Discussion CIB Crystals

• What are some possible techniques that could be
  used to obtain CIB crystals?
• What about the cysteines of CIB?
• Why is it important that the radius of CIB is
  smaller when it is bound to calcium?
• CIB contains a hydrophobic patch, how could this
  information change your crystallization
  conditions?