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1、Protein Crystallography,汪 德 强,重庆医科大学医学检验系生物技术教研室感染性疾病分子生物学教育部重点实验室,一、概述 1 历史的回顾 1895年德国物理学家伦琴发现X射线并因此获得1901年首届诺贝尔物理学奖，X射线历经110年跨越3个世纪，由于众多学者在探索X射线性质、应用、仪器等方面的创新性研究，先后有29位物理学家、晶体学家、化学家、分子生物学家等分别获得了物理（7项）、化学（9项）、生理学或医学（3项）总计19项诺贝尔奖。,1912年劳厄获得了X射线通过晶体后产生的衍射斑点图像（劳厄衍射图），证明了X射线的波动性及其波长范围。随后提出了表示原子排列周期与X射线
2、波长间关系的著名的衍射方程（劳厄方程），并成功地解释了晶体衍射的实验结果。英国物理学家布拉格父子、达尔文等人发展了X射线衍射理论，类比光学反射原理提出了表示晶体结构（晶面间距d）、X射线波长（）与衍射方位（）间的关系的布拉格方程，提出了嵌镶晶体、完整晶体和包含有原子热运动诸因素的衍射强度公式，阐明了X射线,通过晶体产生衍射的付里叶变换本质，获得了X射线的连续光谱与取决于阴极材料的特征光谱。康普顿发现了X射线二次散射时引发的波长的变化(康普顿-吴有训散射)而确定了其粒子性质，从而揭示了X射线的波动与粒子二象性。之后，全世界众多的物理实验室相继开展了对X射线的基础研究工作，并逐步拓展为一个多学科交
3、叉研究热点，主要的应用领域包括：矿物学、物理学、有机与无机化学、分子生物学、医药学、金属与材料科学等。,并最终使X射线衍射成为有机分子（特别是生物活性分子）立体结构测定的有力工具，为研究生理活性物质（药物分子）的立体结构、结构改造、结构预测、结构功能关系为目标的有机晶体学科奠定了基础。对于生物大分子的研究，始于30年代中期，贝纳尔和藿奇金开始用X射线衍射方法研究胃蛋白酶的晶体结构，但直到布拉格主持凯文迪实验室后，才使得这一工作取得突破，为创建分子生物学科奠定了基础。1953年沃森和克里克根据X衍射实验数据建立了脱氧核糖核酸(DNA)的双螺旋结构，并因此获得1962年的诺贝尔生理学和医学奖。,肯
4、德鲁和佩卢茨从30年代开始，应用X衍射方法研究肌红蛋白与血红蛋白的晶体结构，历经20多年的艰苦努力，在众多科学家的共同参与下，终于在1960年获得了这两个蛋白质的三维结构，并因此荣获1962年的诺贝尔化学奖。在1957至1967年的10年中，相继用X衍射方法测定了溶菌酶、胰岛素、胰凝乳蛋白酶A、核糖核酸酶、核糖核酸酶S和羧肽酶的高分辨晶体结构。戴森豪菲尔和胡贝尔、米海尔因测定紫色细菌光合作用中心的三维结构而获得1988年的诺贝尔化学奖，形成了新的蛋白质晶体学科与结构分子生物学科。,物理奖（7项 8人）,化学奖（9项 15人）,生理医学奖（3项 6人）,相关学科发展（2项 2人）,2 X射线晶体
5、结构分析,X射线：表示所用的物理源与晶体相互作用的物理效应衍射晶体：表示固体状态下的一种特殊存在形态晶体生长晶体的几何性质对称性衍射信息中的对称性相位计算中的对称性结构分析：两次付里叶变换，完成第二次付里叶变换的数学方法晶体结构描述,LicT mutant(active)H207D/H269D,LicT wt(inactive),Comparison of licT-wt and licT mutant,Graille*and Zhou*et al.2004 van Tilbeurgh et al.EMBO J.2001,Yang et al.EMBO J.2002,Structure-dir
6、ected drug design,An example of Thy1 from Thermotoga maritima Thy1:thymidylate synthase-complementing protein present in archaea,prokaryotes,viruses NOT in eukaryotes,Lesley,SA et al.PNAS;2002,Thy1-FAD-dUMP,Thy1-dUMP-HEPES,PDB Content Growth(2004/08/01),Output from International Structural Genomics
7、ConsortiaContribution from crystallographers,2004/04/13,Output from International Structural Genomics Consortiium Contribution from NMR spectrometrists,2004/04/13,Future orientations of SG1,Reconstruction of multiprotein complexes(based on interactomics)2,Systematically solving the 3-D structures of
8、 membrane proteins(a challenge of novel techniques)3,Systems Biology,Interactomes:1,Yeast two-hybrid2,TAP(tandem affinity purification)3,Mass Spectrometry4,Co-IP(coimmunoprecipitation)5,Phage display,Overexpress the putative protein complex in vivoor Reconstruct it in vitro from the individual prote
9、ins,Solve the 3-D structure by means of X-ray crystallography Cryo-Electron Microscopy Electron crystallography(2D EM)Electron tomography,Systematically Structure the Membrane Proteins:A big challenge!PDB:26,880 structures,updated on 2004/08/24http:/Membrane proteins:81 structures,updated on 2004/06
10、/15,Structural Biology Processes,X射线衍射实验和结构计算过程Fourier变换与Fourier反变换,Gene of interest,Ideal case,Tragic reality,Design multipleconstructs,Study literature and analog/model cases,Evaluate and optimize expression,Small-scale purification,Evaluate proteinquality,Large-scale purification,Screening,Select
11、 expression system(s),Only a few(or one)constructs,New protein withlittle prior knowledge,Sub-optimal expression,Purification,Limited choice ofexpression systems,I.Recombinant protein over-expression and purification,Expression systems:Bacteria systemYeastInsect cellsMammalian cellsCell-free system,
12、Some Vectors for E.coli Expression System,Protein Expression in Yeast,Cloning of target gene to vector,Transform to yeast Pichia pastoris,Selection of recombinant yeast strain,Yeast cell culture for protein production,Protein Expression in Insect Cells,After recombination,Cloning of target gene to p
13、FastBac,Transform to bacteria with Bacmid,Bacmid transfected to insect cells,Virus assembly in insect cells,Viruses infect Insect Cells for protein production,Strains for expression:Sf9,Sf21,Hi5,Transient Expression In Mammalian Cells,293E cell can be cultured in suspension medium,Recombinant plasmi
14、d with target gene,Transfect to 293E cells with PEI,Harvest cells for protein purification,293EBNA1 Cells With GFP Expressing Vector,A,B,Whole cells on plate;Cells in the same plate to A viewed by GFP florescence,Recombinant Proteins Expression In 293EBNA1 Cells,Lanes:1.Protein standard;2.Control wh
15、ole 293E cells;3.GFP expressed 293E cells;4.HCF-1N380 expressed 293E cells;5.HCF-1N16-363 expressed 293E cells.,Recombinant protein 1(lane 4),1 2 3 4 5,14,20,31,45,67,94,Recombinant protein 2(lane 5),GFP(lane3),Cell-free System for Protein Production,Sometimes it can produce soluble protein which ca
16、n not be expressed as soluble form with cellular system.,Roche:Rapid Translation System(RTS),Rapid protein expression,Toxic protein expression,ProteinProtein Complex Expression and Purification:a.Proteins express separately;b.Proteins co-express in one cell.2.Protein-Nucleic Acid：a.Protein-DNA Compl
17、ex；b.Protein-RNA Complex.,Producing Protein Complexes for Crystallization,Methods for production of recombinant protein complexes by in vivo reconstitution in E.coli1.Use compatible vectors,such as pMR101(p15A ori)and pET15B(pBR322 ori);2.Use one vector with more than one expression cassettes-polyci
18、stronic；Benefits of in vivo reconstitution(coexpression)efficiencyone round of expressionone round of purificationqualitycoexpression and cofolding of polypeptides in the presence of cellular chaperones may increase yield of functional complex,ProteinProtein Complex Expression and Purification,Prote
19、inDNA Complex,Protein solubility:higher in high salt buffer usually;Protein-DNA complex stability:more stable than protein alone;DNA length and sequence used for crystallization:a.additional base pairs;b.sticky ends;4.Purification of DNA oligos:HPLC with hydrophobic interaction,C4 etc;5.Trapping rea
20、ction intermediate:disulfide bridge;protein point mutation,etc;6.Preparation of protein-DNA complexes:mix with extra molar DNA;Crystallization:PEG or MPD in low slat buffer;Example:over 6000 trial for protein-DNA complex.,Protein-RNA Complex,Difficulties:avoid of RNase!1.Phosphate groups interfere c
21、rystal packing;2.Elongated RNAs pack loosely;RNA engineering:blunt or sticky ends;deletion,replacement,etc;RNA preparation:1.Synthesis;2.In vitro transcription;,Protein Modification for Crystallization,1.Protein inhibitor,partner and monoclonal antibody;2.Protein post-translational modification;3.Pr
22、otein mutagenesis:truncation,mutation,deletion,Protein Mutagenesis,1.Truncation or deletion:secondary structure prediction;DXMS result;homologue protein sequences comparison or structure comparison;Mutation methods a.Selected point mutation;b.Random mutation：DNA shuffling for chimeric protein;random
23、 mutation by low-fidelity PCR.,Hydrogen/deuterium exchange mass spectroscopy(DXMS)for protein analysis,Keenan,Robert J.et al.(2005)Proc.Natl.Acad.Sci.USA 102,8887-8892,Random mutation by DNA shuffling,Mutation selection by GFP folding reporter,GFP,Target Protein,Correct Folding of Target Protein,Mis
24、folding of Target Protein,Fluroscence,NH3+,COO-,No Fluroscence,(Waldo,GS.Et al.1999,Nature Biotech.17:691),GFP Folding Reporter,Wild-type gene,Random mutagenesis with Polymerase(Exo-),Random Mutagenesis.Clone into GFP vector.Select the brightest colonies.Test the solubility of Kelch-GFP.Reclone into
25、 GST fusion vector.Test the solubility of GST-Kelch.,PCR,Protein purification method,Affinity Column:by tags or antibodies;Ion exchange column;Size exclusion column;Hydrophobic interaction;others,Metal affinity or other affinity columns TCEP is a very good alternative to DTT or BME when you must hav
26、e a reducing agent during purification.Most proteins will bind to Q resins at pH 7.0-8.5.Check if DEAE can be used since its purification factor is much higher.Lower pH results in higher purification factor as long as target protein still binds.DNA-binding proteins often ride on the bound DNA and el
27、ute at moderate ionic strength.DNA precipitation(e.g.via polyethyleneimine addition)is a useful,but somewhat risky step.Most proteins do not bind to S resins at pH 7.0-8.5.Majority will still not bind at pH 6.0-7.0,therefore an S column at pH 6.0-8.0 has a very good purification factor if target pro
28、tein is bound.A CM-column,Optimize protein purification,has an even higher purification factor.Virtually no proteins bind to CM columns at pH 8.0.The use of acidic columns may require passing through the pI of target protein.Hydroxyapatite can give very high purification factors.Size-exclusion chrom
29、atography is very useful and normally non-damaging method.Purification by protein properties,Optimize gene or expression,Apparent problem,Misfolding,Low rate of synthesis,Protein degradation,Expression system,Fusionsor tags,Promoters,Expression conditions,Codon bias,Co-expression,Domain structure,Po
30、ssible changes,Misfolding,Folding efficiencyLack of proper chaperones.Synthesis rateSynthesis is too fast for the folding capacity of the system.Protein localizationProtein requires specific compartmentalization(i.e.periplasmic or intramembrane)to fold.Post-translational modificationEukaryotic prote
31、ins often require specific PTM to mature.,Folding efficiencyToxic proteins are often dominant-negative.As a result,the worse is the folding of such proteins,the more(incompetent)protein is actually made.Synthesis rateSynthesis can be negatively affected by initiation rate,codon bias,no proper nutrie
32、nts or low-level co-factors(e.g.certain metal ions).Protein localizationProtein is translocated directly into a specific compartment(i.e.periplasmic or intramembrane).As a result,if the compartment is not available,the ribosomes stall or abort.,Low rate of synthesis,Folding efficiencyIf inclusion bo
33、dies are not formed,improperly folded protein can be rapidly degraded.Synthesis rateLow rate of synthesis can result in the need for longer growth times and therefore longer exposure of the protein to proteases.Protein localizationProtein compartmentalization can have significant effect on degradati
34、on,e.g.when protein is subjected to signal peptidases in bacterial periplasm.Post-translational modificationEukaryotic systems use ubiquitinylation as degradation signal.Membrane-associated proteases can specifically attack proteins that bear membrane-association or transmembrane signals.,Protein de
35、gradation,Fusions or tagsCan have a tremendous negative or positive effect on foldingCo-expressionCan be very helpful Expression conditionsLowering the temperature often results in more folded protein.Functional expression can also be regulated through nutrients and co-factors.Domain structureProper
36、 definition of domain boundaries can have paramount effect on folding.,Expression system,Fusionsor tags,Expression conditions,Foldingefficiency,Co-expression,Domain structure,Expression systemIt is easier(and cheaper)to produce massive quantities of proteins in bacteria or yeast.PromotersExpression
37、conditionsTemperature,nutrient/oxygen content,antibiotics,etc.Domain structureTranslational interdomain pausing can slow down the overall process or result in abortive expression.Codon biasCodon optimization ensures that rare codons do not cause translational pausing or abortion.,Expression system,F
38、usionsor tags,Expression conditions,Synthesisrate,Promoters,Domain structure,Codon bias,Avoid freeze-thaw cycles.Most proteins do not tolerate freeze-drying or prolonged storage at 4C.Storage some proteins in 30-50%glycerol or ethylene glycol at 20C or 80C is a useful alternative.Flash-freezing prot
39、ein stock in small aliquots.,Optimize existing sample properties,II.Protein Crystallization,General approach for protein crystallization,Macromolecular crystals are composed of approximately 50%solvent on average,though this may vary from 25 to 90%depending on the particular macromolecule.Macromolec
40、ular crystal growth is still largely empirical in nature.It is still a mystery for the reasons that some proteins could not be crystallized.Searching systematically and broadly;,Crystal screening,Crystal optimization,Two steps for protein crystal obtaining,Screening,Robotic,Manual,Cheap Time-tested
41、Readily availableAllows for creativity,Multitude of conditions Highly reproducible Easy to document and track data Lower consumption of protein,1.Altering the protein itself:such as change of pH to alter protein ionic surface;2.By altering the chemical activity of the water:e.g.,by addition of salt;
42、3.By altering the degree of attraction of one protein molecule for another:e.g.,change of pH,addition of bridging ions;4.Altering the nature of the interactions between the protein molecules and the solvent:e.g.,addition of polymers or ions.,Crystallization of a macromolecule absolutely requires the
43、 creation of a supersaturated state.,Methods for creating supersaturation,Table 1.Methods for creating supersaturation,Table 2.Methods for promoting a solubility minimum,Precipitants used in macromolecular crystallization,1.Salts:(NH4)2SO4 2.Volatile organic solvents:Ethanol3.Long chain polymers:PEG
44、40004.Low molecular weight polymers and non-volatile organic compounds:MPD,Table 3.Precipitants used in macromolecular crystallization,Factors affecting crystallization,For macromolecule:purity,stability,modification,etc;Some chemical factors:pH,precipitant,ion strength,specific ions,etc;Some physic
45、al factors:temperature,crystallization method,time,etc.,Table4.Factors affecting crystallization,Homogeity:purity is very important;Solubility:dissolve protein to high concentration;Stability:maintain protein as stable as possible;Supersaturation:alter the properties of solution for supersaturation;
46、Association:try to promote ordered association;Nucleation:try to promote the formation of nuclei;Variety:try as many methods as possible;Liquid impurities:avoid impurities in the mother liquid;Preservation:protect plate and crystal from shock and disruption;,Conclusions:Some important principles,Optimize geneor expression,Optimize existingsample properties,Optimize proteinpurification,Bad screening result,Look for homologs,Protein Crystal Diffraction,Thanks,
