Presentation on theme: "Growing Protein Crystals"— Presentation transcript:
1 Growing Protein Crystals Using Calcium-Integrin Binding Protein as a Model Presented byChad BlameyFBPxtals/xtals.html
2 Goals What are good crystals Understand how crystals grow Why getting good crystals is importantUnderstand how crystals growDiscus techniques for crystallizing proteinsApplication type of discussionStrategies for optimizing crystal growthUnderstanding your favorite proteinCIB as a modelMy favorite protein!!!Lysozyme demonstration
3 Lysozyme Demonstration Buffer30% w/v Polyethelene glycol 50001 M NaCl50 mM NaAcetate pH 4.5Lysozyme Protein100mg/ml50 mM Na Acetate pH 4.52l4lglass slideShould make large crystals in less than 15 minutes.We will watch it for the hour of the lecture.
4 Everyone Should KnowProtein crystals are precipitated protein in solutionsYou can think of them highly concentrated aqueous solutions (usually about 500 mg/ml)Amorphous precipitation is randomCrystals are orderedThis is the property we are interested inFigure 3.1 CMCCGray areas!CrystalsCrystallinePrecipitation
5 Crystallization: Needs Obtaining quality crystals is by far the limiting step to solving a structureCrystals need to be of sufficient size and quality to diffract x-raysSize: Normally should be 100 m in smallest dimensionQuality: 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 qualityGrowing good crystals is key to a good structure
6 CrystallizationWith enzymes is is often important to maintain enzymatic activity in crystalSome enzymes can function in crystalBest way to test crystal quality is by mounting a crystal and attempting to diffract x-raysVisual inspection helpful tooMay 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 spots9 Å4.5 Å
8 Higher order visible (circle) Good vs Poor DataCa+007M0354.5 ÅPoor, smeary spotsNotice ‘twined’ spots4.1 ÅGood! Round spotsHigher order visible (circle)
9 Do spots match mathematical predictions? Spot PredictionCrystal M035Do spots match mathematical predictions?
10 How Do Proteins Crystallize? For crystallization to occur it has to be thermodynamically favorablePrecipitants remove available water forcing proteins to associate with each otherHopefully in a organized fashionWater++Protein+precipitant+----polyethelyene glycolsaltssugarsorganic solvents
11 Growing Crystals: Hanging Drop Method Widely usedVapor diffusionDrop equalizes with reservoirVolume of drop slowly decreasesProtein concentration slowly increasesCMCC Figure 3.2crystalsdropreservoirThis method relies on vapor diffusion so that the soulition in the drop above becomes roughly equivalent to the large reservoir below.[X][Y]Sitting drop
12 Phases of Proteins In Solution Not to be confused with phases of light[Protein]SolubilitySupersaturationMetastabileUndersaturatedPrecipitationCrystalsGrowth & NucleationGrowth onlyBarrier of NucleationSoluble proteinFigure 3.3Crystals may grow in the metastable region if somehow nuclei are present. Nucleation will only occur spontaneously in the supersaturated region. The line separating one phase form the other are actually probabilities and do not exist in realityCMCC figure 3.3
13 Nucleation & Growth Basic concept: Phase diagram [Protein]SolubilitySupersaturationMetastabileUndersaturatedFigure 3.3Phase diagramConcentrate solution enough so nucleation occurs in only a few casesInitial growth pulls some protein out of solutionReducing [protein] back into metastable rangeGrow only a few large crystals
14 Optimize Crystal Growth The number of factors can be overwhelmingFocus on those factors which most effect growthSet up arrays to vary two different conditions at onceCross your fingers[X][Y]
15 The Tricky PartConditions for crystallization are dependent on each-otherCrystal quality will change as you vary growth conditionsFigure 3.4[B][A]For solution made up of three parts A, B and C.Changing [C] will effect the quality of the crystal interms of [A].
16 Growing Crystals: Other Techniques Spin0 hours6 hours12 hoursUlatacentrifugationSpin at extremely high speeds, hundreds of thousands of g’sSlowly increases the relative protein concentrationDialysisUses liquid-liquid diffusionDiffusion is slowRate controlled by membrane
17 Crystal ScreensHampton Research screen tests a wide assortment of conditions of salts, buffers, pH’s and additivesBest conditions from literatureOften first hits with screens are small poor quality crystalsDo not use the absence of crystals as a gauge of conditions rather use solubility
18 Factors Effecting Crystal Growth *Most importantIonic Strength*Specific Ions (Ca2+)Protein Concentration*DetergentsInorganic PrecipitantpH*Temperature*TimeMonodispersion*VibrationsPressureGravityRelative Proportion of ConditionsPurity Of Protein*Access to water*LigandsBinding partners
19 Characterization of Protein Of course the more you know about your protein the easier it is to manipulateCystine is often the most critical a.a.CIB has three and no disulfide bonds, but cause multimersKey ligands and metals, like Ca (for CIB)Stability in certain solutionsHydrodynamic radius (NMR)Stability (CD is great)Dynamic light scatteringMass spec
20 CIB Protein Characterization IEFWestern-BlotCIB purified w/o reducing agentsSDS-PAGENo DTTDTTMarker22CIBpH 5.6CIB purified w/ reducing agentsFurther characterization of a protein canimprove purity and therefore crystal quality
21 Ligands and Co-crystallization Try to obtain crystals with different ligands and/or co-crystallize with another proteinMetals, peptides & binding proteinsProteins with a known structure can simplify the processEnzymes and Substrate complexesNon-competitive inhibitorsSubstrate analogsOften changes protein conformationTwo structures!This gives information about how the protein function
22 AdditivesOften designed to reduce strength of protein-protein interactionDetergents important categoryReducing agentsOrganic solvents
23 Small CrystalsOften small crystals can be made larger by microseeding new drops with previously grown crystals or adding more protein solutionMultinucleation can be avoided by reducing the temperature or adding glycerolCrystals only need to be large enough to diffract x-rays well
24 Radical ApproachesRemove either N- or C-terminus by weak proteolysis or by molecular cloningOften termini can be disordered which interferes with lattice formationCrystallize with a fusion proteinFusion proteins are well documented with a solved structure that easily from a lattice, example: GST“Pull” the fusion protein into an ordered crystalCan use the protein for molecular replacement to solve phaseMany 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 technologyDomains can be crystallized separatelyIssues:Different conformations from the native state likelyDomains can only be part of the storyChanging the means starting over in terms of crystallization solution
26 DiscussionGiven 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 LysozymeVapor 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 DiscussionWhat 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?