Presentation on theme: "Growing Protein Crystals Using Calcium-Integrin Binding Protein as a Model Presented by Chad Blamey FBP www.scripps.edu/~arvai/ xtals/xtals.html."— Presentation transcript:
Growing Protein Crystals Using Calcium-Integrin Binding Protein as a Model Presented by Chad Blamey FBP www.scripps.edu/~arvai/ xtals/xtals.html
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
Lysozyme Demonstration 2l2l 2l2l 4l4l 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.
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! CrystalsPrecipitation Crystalline
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
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
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
Good vs Poor Data Ca+007M035 4.5 Å Poor, smeary spots Notice ‘twined’ spots 4.1 Å Good! Round spots Higher order visible (circle)
Spot Prediction Crystal M035 Do spots match mathematical predictions?
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 polyethelyene glycol salts sugars organic solvents precipitant - - - - + + + +
Growing Crystals: Hanging Drop Method Widely used Vapor diffusion Drop equalizes with reservoir Volume of drop slowly decreases Protein concentration slowly increases CMCC Figure 3.2 [X] [Y] Sitting drop reservoir drop crystals
Phases of Proteins In Solution Not to be confused with phases of light [Protein] Solubility SupersaturationMetastabile Undersaturated Precipitation Crystals Growth & Nucleation Growth only Barrier of Nucleation Soluble protein Figure 3.3 CMCC figure 3.3
[Protein] Solubility SupersaturationMetastabile Undersaturated Figure 3.3 Phase diagram1.Concentrate solution enough so nucleation occurs in only a few cases 2.Initial growth pulls some protein out of solution 3.Reducing [protein] back into metastable range 4.Grow only a few large crystals Basic concept: Nucleation & Growth
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 [X] [Y]
The Tricky Part Conditions for crystallization are dependent on each- other Crystal quality will change as you vary growth conditions Figure 3.4 [A] [B] For solution made up of three parts A, B and C. Changing [C] will effect the quality of the crystal in terms of [A].
Growing Crystals: Other Techniques Spin at extremely high speeds, hundreds of thousands of g’s Slowly increases the relative protein concentration Spin 0 hours 6 hours12 hours Ulatacentrifugation Dialysis Uses liquid-liquid diffusion Diffusion is slow Rate controlled by membrane
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
Factors Effecting Crystal Growth Ionic Strength* Specific Ions (Ca 2+ ) 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 * Most important
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
CIB Protein Characterization IEF Western- Blot IEF CIB purified w/o reducing agents SDS-PAGE No DTTDTT Marker 22 CIB pH 5.6 CIB purified w/ reducing agents Further characterization of a protein can improve purity and therefore crystal quality
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
Additives Often designed to reduce strength of protein-protein interaction Detergents important category Reducing agents Organic solvents
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
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
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
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?
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?
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
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?