Growing Protein Crystals

1. Growing Protein Crystals Using Calcium-Integrin Binding Protein as a ModelPresented by Chad Blamey FBP www.scripps.edu/~arvai/ xtals/xtals.html

2. Goals • What are good crystals • Why getting good crystals is important • Understand how crystals grow • Discus techniques for crystallizing proteins • Application type of discussion • Strategies for optimizing crystal growth • Understanding your favorite protein • CIB as a model • My favorite protein!!! • Lysozyme demonstration

3. 2l 2l 4l Lysozyme Demonstration Buffer 30% w/v Polyethelene glycol 5000 1 M NaCl 50 mM NaAcetate pH 4.5 Lysozyme Protein 100mg/ml 50 mM Na Acetate pH 4.5 glass slide Should make large crystals in less than 15 minutes. We will watch it for the hour of the lecture.

4. Everyone Should Know • Protein crystals are precipitated protein in solutions • You can think of them highly concentrated aqueous solutions (usually about 500 mg/ml) • Amorphous precipitation is random • Crystals are ordered • This is the property we are interested in • Figure 3.1 CMCC • Gray areas! Crystals Precipitation Crystalline

5. Crystallization: Needs • Obtaining quality crystals is by far the limiting step to solving a structure • 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

6. Crystallization • With enzymes is is often important to maintain enzymatic activity in crystal • Some enzymes can function in crystal • Best way to test crystal quality is by mounting a crystal and attempting to diffract x-rays • Visual inspection helpful too • May not be meaningful

7. Low vs High Data • Difference between 9.0 Å and 4.5 Å • The higher the resolution the better! • CIB crystal spots 9 Å 4.5 Å

8. Good vs Poor Data Ca+007 M035 4.5 Å Poor, smeary spots Notice ‘twined’ spots 4.1 Å Good! Round spots Higher order visible (circle)

9. Spot Prediction Crystal M035 Do spots match mathematical predictions?

10. How Do Proteins Crystallize? • For crystallization to occur it has to be thermodynamically favorable • Precipitants remove available water forcing proteins to associate with each other • Hopefully in a organized fashion Water + + Protein + precipitant + - - - - polyethelyene glycol salts sugars organic solvents

11. [X] [Y] Growing Crystals: Hanging Drop Method crystals • Widely used • Vapor diffusion • Drop equalizes with reservoir • Volume of drop slowly decreases • Protein concentration slowly increases • CMCC Figure 3.2 drop reservoir Sitting drop

12. Crystals Figure 3.3 Precipitation Metastabile Supersaturation Growth & Nucleation Solubility Barrier of Nucleation Undersaturated Growth only [Protein] Soluble protein Phases of Proteins In Solution Not to be confused with phases of light CMCC figure 3.3

13. Phase diagram Figure 3.3 Metastabile Supersaturation Solubility Undersaturated [Protein] 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

14. [X] [Y] 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

15. The Tricky Part • Conditions for crystallization are dependent on each-other • Crystal quality will change as you vary growth conditions • Figure 3.4 [B] [A] For solution made up of three parts A, B and C. Changing [C] will effect the quality of the crystal in terms of [A].

16. Spin 6 hours 12 hours 0 hours Growing Crystals:Other Techniques Ulatacentrifugation • Spin at extremely high speeds, hundreds of thousands of g’s • Slowly increases the relative protein concentration Dialysis • Uses liquid-liquid diffusion • Diffusion is slow • Rate controlled by membrane

17. Crystal Screens • Hampton Research screen tests a wide assortment of conditions of salts, buffers, pH’s 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

18. Ionic Strength* Specific Ions (Ca2+) 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 Factors Effecting Crystal Growth *Most important

19. Characterization of Protein • Of course the more you know about your protein the easier it is to manipulate • Cystine is often the most critical a.a. • CIB has three and no disulfide bonds, but cause multimers • Key ligands and metals, like Ca (for CIB) • Stability in certain solutions • Hydrodynamic radius (NMR) • Stability (CD is great) • Dynamic light scattering • Mass spec

20. CIB purified w/ reducing agents CIB purified w/o reducing agents IEF Western- Blot IEF SDS-PAGE Marker No DTT DTT CIB pH 5.6 22 CIB Protein Characterization Further characterization of a protein can improve purity and therefore crystal quality

21. Ligands and Co-crystallization • Try to obtain crystals with different ligands and/or co-crystallize with another protein • Metals, peptides & binding proteins • Proteins with a known structure can simplify the process • Enzymes and Substrate complexes • Non-competitive inhibitors • Substrate analogs • Often changes protein conformation • Two structures! • This gives information about how the protein function

22. Additives • Often designed to reduce strength of protein-protein interaction • Detergents important category • Reducing agents • Organic solvents

23. 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

24. 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, example: GST • “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

25. Radical Approaches, Cont • Mutants: • Specific residues problem residues can be mutated using recombinant DNA technology • Domains can be crystallized separately • Issues: • Different conformations from the native state likely • Domains can only be part of the story • Changing the means starting over in terms of crystallization solution

26. Discussion • Given a hypothetical protein that doesn’t give you any positive hits in your first screen what could you do to obtain quality crystals? • Meaning: almost no precipitation in each drop! • Likewise what if you get lots of precipitation? • 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?

27. Lysozyme • Vapor diffusion takes about 12 hours to complete. Where on phase diagram did the [lysozyme] start given almost no vapor diffusion occurred? • Lysozyme in other solutions crystallizes much more slowly (period of days). Which solutions would yield higher quality crystals? Why? • What are some of the properties of lysozyme that allow it to crystallize so quickly?

28. CIB • Similar to a ubiquitous protein Calmodulin • Binds calcium • Regulates other proteins (13 so far) • Found in most tissues types: brain, muscle etc. • No enzymatic activity

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