蛋白质晶体学课件

1、蛋白质晶体学PPT课件Protein Protein Crystallography Crystallography 汪汪 德德 强强感染性疾病分子生物学教育部重点实验室感染性疾病分子生物学教育部重点实验室蛋白质晶体学PPT课件一、概述一、概述 1 1 历史的回顾历史的回顾 1895年德国物理学家伦琴发现年德国物理学家伦琴发现X射线并因射线并因此获得此获得1901年首届诺贝尔物理学奖，年首届诺贝尔物理学奖，X射线历射线历经经110年跨越年跨越3个世纪，由于众多学者在探索个世纪，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，射线性质、应用、仪器等方面的创新性研究，先后有先后有29位

2、物理学家、晶体学家、化学家、分位物理学家、晶体学家、化学家、分子生物学家等分别获得了物理（子生物学家等分别获得了物理（7项）、化学项）、化学（9项）、生理学或医学（项）、生理学或医学（3项）总计项）总计19项诺贝项诺贝尔奖。尔奖。蛋白质晶体学PPT课件 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（

3、）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射线蛋白质晶体学PPT课件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。蛋白质晶体学PPT课件 并最终使并最终使X X射线衍射成为有机分子（特别射线衍射

4、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，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。为目标的有机晶体学科奠定了基础。 对于对于生物大分子的研究生物大分子的研究，始于始于3030年代中年代中期期，贝纳尔和藿奇金开始用，贝纳尔和藿奇金开始用X X射线衍射方法研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，凯文迪实验室后，才使

5、得这一工作取得突破，为创建为创建分子生物学科分子生物学科奠定了基础。奠定了基础。 1953 1953年沃森和克里克根据年沃森和克里克根据X X衍射实验数据衍射实验数据建立了脱氧核糖核酸建立了脱氧核糖核酸( (DNA)DNA)的双螺旋结构，并的双螺旋结构，并因此获得因此获得19621962年的诺贝尔生理学和医学奖。年的诺贝尔生理学和医学奖。蛋白质晶体学PPT课件 肯德鲁和佩卢茨从肯德鲁和佩卢茨从30年代开始，应用年代开始，应用X衍衍射方法研究射方法研究肌红蛋白肌红蛋白与与血红蛋白血红蛋白的晶体结构，的晶体结构，历经历经20多年的艰苦努力，在众多科学家的共多年的艰苦努力，在众多科学家的共同参与下，

6、终于在同参与下，终于在1960年获得了这两个蛋白年获得了这两个蛋白质的三维结构，并因此荣获质的三维结构，并因此荣获1962年的诺贝尔年的诺贝尔化学奖。化学奖。 在在1957至至1967年的年的10年中，相继用年中，相继用X衍衍射方法测定了射方法测定了溶菌酶溶菌酶、胰岛素胰岛素、胰凝乳蛋白胰凝乳蛋白酶酶A、核糖核酸酶核糖核酸酶、核糖核酸酶核糖核酸酶S和和羧肽酶羧肽酶的的高分辨晶体结构。高分辨晶体结构。 戴森豪菲尔和胡贝尔、米海尔因测定戴森豪菲尔和胡贝尔、米海尔因测定紫紫色细菌光合作用中心色细菌光合作用中心的三维结构而获得的三维结构而获得1988年的诺贝尔化学奖，形成了新的年的诺贝尔化学奖，形成了

7、新的蛋白质晶体蛋白质晶体学科学科与与结构分子生物学科结构分子生物学科。蛋白质晶体学PPT课件 物理奖 （7项 8人） 成成 就就(另一发现者Mosley因第一次世界大战阵亡而未获奖)年代获奖者成 就1901Wilhelm Konrad Rntgen (德)W.K.伦琴1895年发现X射线及其性质1914Max Von Laue(德) M.J.劳厄1912年发现晶体X射线衍射1915William Henry Bragg(英)W.H.布拉格1912年建立X射线衍射晶体结构分析William Lawrence Bragg(英)W.L.布拉格1917Charles Glouer Barkla(英)C

8、.G.巴克拉1909年建立X射线光谱学的K、L系(另一发现者Mosley因第一次世界大战阵亡而未获奖)1924Karl Manne Georg Siegbahn(瑞典)K.M.G.西格班1912年发现X射线光谱学的M系1927Arthur Holly Compton(美)A.H.康普顿1919年发现X射线能量变化的康普顿效应1981Kai M. Siegbahn(瑞典)K.西格班1956年发现X射线光电子能谱蛋白质晶体学PPT课件 化学奖 （9项 15人）1936Peter Debye(荷）P.德拜1916年 提出粉末X射线晶体结构分析1946James Batchelle Sumner(美)

9、J.B.萨姆纳1926年获得尿素酶结晶；1937年获得氧化氢酶结晶等John Noward Northrop(美)J.N.诺思罗普1930年获得胃蛋白酶结晶等Wendell Stanley(美)W.斯坦利1935年获得菸草花叶病毒(TMV)结晶；1946年获得流感病毒结晶1954Linus Pauling(美)L.鲍林用X射线衍射法确定多肽结构的化学键本质1962John Cowdery Kendrew(英)J.C.肯德鲁用X射线衍射法测定肌红蛋白结构Max Ferdinand Peruty(英)M.F.佩鲁茨用X射线衍射法测定血红蛋白结构1964Dorothy Mary Cronfoot H

10、odgkin(英)D.M.C.霍奇金用X射线衍射法测定青霉素与B12分子结构1976William Nunn Lipscomb(美)W.N.利普斯科姆低温X射线衍射法确定硼氢化合物分子结构1982Aaron Klug(英)A.克卢格蛋白质分子结构的电子显微镜三维重组1985Herbert A. Hauptman(美)H.A.豪普特曼X射线衍射分析直接法的建立Jerome Karle(美)J.卡尔1988Hartmut Michel(德)H.米歇尔以X射线衍射法测定了细菌光合作用反应中心的分子立体结构Johann Deisenhifer(德)J.戴森霍菲尔Robert Huber(德)R.胡贝尔

11、蛋白质晶体学PPT课件 生理医学奖 （3项 6人）年代年代获奖者获奖者成成 就就1946Hermann Joseph Muller(美)H.J.缪勒发现X射线照射引起基因突变，建立了辐射遗传学1962Francis Harry Compton Crick(英)F.H.C.克里克1953年应用X射线衍射法建立了DNA分子结构模型James Dewey Watson(英)J.D.沃森Maurice Hugh Frederick Wilkins(英)M.H.F.威尔金斯1979Allan M. Cormack(美)A.M.科马克1969年建立了计算机辅助X射线断层扫描(CT)Godfrey N. H

12、ounsfield(英)G.N.豪斯菲尔德1937Clinton Joseph Davisson(美)C.J.戴维森发现电子衍射技术1994Clifford Clenwood Shull(美)C.C.沙尔发现中子衍射技术蛋白质晶体学PPT课件2 X射线晶体结构分析l 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l 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l 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蛋白质晶体学PPT课件LicT mutant (active)H207D/H269DLicT wt

13、(inactive)Comparison of licT-wt and licT mutantGraille* and Zhou* et al. 2004 van Tilbeurgh et al. EMBO J. 2001蛋白质晶体学PPT课件Yang et al. EMBO J. 20021122mRNA1122mRNAPKD=10M KD=1MCATPRD2PRD1RATCATRNA蛋白质晶体学PPT课件 Structure-directed drug design An example of Thy1 from Thermotoga maritima Thy1: thymidylate

14、synthase-complementing protein present in archaea, prokaryotes, viruses NOT in eukaryotesLesley, SA et al. PNAS; 2002蛋白质晶体学PPT课件Thy1-FAD-dUMP Thy1-dUMP-HEPES 蛋白质晶体学PPT课件PDB Content Growth (2004/08/01)蛋白质晶体学PPT课件蛋白质晶体学PPT课件52,66227,99916,0975,816 5,6992,133 1,031892645010,00020,00030,00040,00050,0006

15、0,000targetsclonedexpressedsolublepurifiedcrystallizeddiffraction-qual.diffractionstructuresOutput from International Structural Genomics ConsortiaContribution from crystallographers, 2004/04/13蛋白质晶体学PPT课件2,75583824920005001,0001,5002,0002,5003,000targetsHSQCNMR assignedNMR structuresOutput from Int

16、ernational Structural Genomics Consortiium Contribution from NMR spectrometrists, 2004/04/13蛋白质晶体学PPT课件Future orientations of SG1, Reconstruction of multiprotein complexes (based on interactomics)2, Systematically solving the 3-D structures of membrane proteins (a challenge of novel techniques)3, Sy

17、stems Biology 蛋白质晶体学PPT课件Interactomes:1, Yeast two-hybrid2, TAP (tandem affinity purification)3, Mass Spectrometry4, Co-IP (coimmunoprecipitation)5, Phage display蛋白质晶体学PPT课件Overexpress the putative protein complex in vivoor Reconstruct it in vitro from the individual proteins Solve the 3-D structure

18、 by means of X-ray crystallography Cryo-Electron Microscopy Electron crystallography (2D EM) Electron tomography蛋白质晶体学PPT课件Systematically Structure the Membrane Proteins: A big challenge!PDB: 26,880 structures, updated on 2004/08/24/pdb/index.html Membrane proteins: 81 structures,

19、updated on 2004/06/15 http:/www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html蛋白质晶体学PPT课件Structural Biology ProcessesStructural Biology Processes蛋白质晶体学PPT课件 X射线衍射实验和结构计算过程Fourier变换与Fourier反变换蛋白质晶体学PPT课件Gene of interestIdeal caseIdeal caseTragic realityTragic realityDesign multipleconstructs

20、Study literature and analog/model casesEvaluate and optimize expressionSmall-scale purificationEvaluate proteinqualityLarge-scale purificationScreeningSelect expression system(s)Only a few (or one)constructsNew protein withlittle prior knowledgeSub-optimal expressionPurificationLimited choice ofexpr

21、ession systems蛋白质晶体学PPT课件I. Recombinant protein over-I. Recombinant protein over-expression and purificationexpression and purificationExpression systems: Bacteria system Yeast Insect cells Mammalian cells1. Cell-free system蛋白质晶体学PPT课件Some Vectors for E.coli Expression System蛋白质晶体学PPT课件Protein Expre

22、ssion in YeastCloning of target gene to vectorTransform to yeast Pichia pastorisSelection of recombinant yeast strainYeast cell culture for protein production蛋白质晶体学PPT课件Protein Expression in Insect CellsAfter recombinationCloning of target gene to pFastBacTransform to bacteria with BacmidBacmid tran

23、sfected to insect cellsVirus assembly in insect cellsViruses infect Insect Cells for protein productionStrains for expression: Sf9, Sf21, Hi5蛋白质晶体学PPT课件Transient Expression In Mammalian Cells293E cell can be cultured in suspension medium Recombinant plasmid with target geneTransfect to 293E cells wi

24、th PEIHarvest cells for protein purification蛋白质晶体学PPT课件293EBNA1 Cells With GFP Expressing VectorABWhole cells on plate; Cells in the same plate to A viewed by GFP florescence蛋白质晶体学PPT课件Recombinant Proteins Expression In 293EBNA1 Cells Lanes: 1. Protein standard; 2. Control whole 293E cells; 3. GFP e

25、xpressed 293E cells; 4. HCF-1N380 expressed 293E cells; 5. HCF-1N16-363 expressed 293E cells. Recombinant protein 1 (lane 4)1 2 3 4 5142031456794Recombinant protein 2 (lane 5)GFP (lane3)蛋白质晶体学PPT课件Cell-free System for Protein ProductionSometimes it can produce soluble protein which can not be expres

26、sed as soluble form with cellular system.Roche: Rapid Translation System (RTS) Rapid protein expressionToxic protein expression蛋白质晶体学PPT课件ProteinProtein Complex Expression and Purification: a. Proteins express separately; b. Proteins co-express in one cell.2. Protein-Nucleic Acid： a. Protein-DNA Com

27、plex； b. Protein-RNA Complex.Producing Protein Complexes Producing Protein Complexes for Crystallizationfor Crystallization蛋白质晶体学PPT课件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)

28、;2. Use one vector with more than one expression cassettes-polycistronic；Benefits of in vivo reconstitution (coexpression)efficiency one round of expression one round of purificationquality coexpression and cofolding of polypeptides in the presence of cellular chaperones may increase yield of functi

29、onal complexProteinProtein Complex Expression and Purification蛋白质晶体学PPT课件ProteinProteinDNADNA ComplexComplexProtein 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;

30、 b. sticky ends;4. Purification of DNA oligos: HPLC with hydrophobic interaction, C4 etc;5. Trapping reaction 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 600

31、0 trial for protein-DNA complex.蛋白质晶体学PPT课件Protein-RNA ComplexDifficulties: avoid of RNase! 1. Phosphate groups interfere crystal packing; 2. Elongated RNAs pack loosely;RNA engineering: blunt or sticky ends; deletion, replacement, etc;RNA preparation: 1. Synthesis; 2. In vitro transcription;蛋白质晶体学P

32、PT课件Protein Modification for Crystallization1. Protein inhibitor, partner and monoclonal antibody;2. Protein post-translational modification;3. Protein mutagenesis: truncation, mutation, deletion蛋白质晶体学PPT课件Protein Mutagenesis1. Truncation or deletion: secondary structure prediction; DXMS result; hom

33、ologue protein sequences comparison or structure comparison;Mutation methods a. Selected point mutation; b. Random mutation：DNA shuffling for chimeric protein; random mutation by low-fidelity PCR.蛋白质晶体学PPT课件Hydrogen/deuterium exchange mass spectroscopyHydrogen/deuterium exchange mass spectroscopy (D

34、XMS) for protein analysis(DXMS) for protein analysis 蛋白质晶体学PPT课件Keenan, Robert J. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 8887-8892Random mutation by DNA Random mutation by DNA shufflingshuffling 蛋白质晶体学PPT课件Mutation selection by GFP folding reporter GFPTarget ProteinCorrect Folding of Target P

35、roteinMisfolding of Target ProteinFluroscenceNH3+COO-No Fluroscence(Waldo, GS. Et al.1999,Nature Biotech.17:691)蛋白质晶体学PPT课件GFP Folding ReporterGFP Folding Reporter GFPTargetpGFPuvWild-type geneRandom mutagenesis with Polymerase(Exo-)-Random Mutagenesis.-Clone into GFP vector.-Select the brightest co

36、lonies.-Test the solubility of Kelch-GFP.-Reclone into GST fusion vector.-Test the solubility of GST-Kelch.PCR蛋白质晶体学PPT课件Protein purification methodProtein purification methodAffinity Column: by tags or antibodies;Ion exchange column;Size exclusion column;Hydrophobic interaction;others蛋白质晶体学PPT课件Met

37、al affinity or other affinity columns TCEP is a very good alternative to DTT or BME when you must have 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 purificat

38、ion factor as long as target protein still binds. DNA-binding proteins often ride on the bound DNA and elute 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 sti

39、ll 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 protein is bound. A CM-columnOptimize protein purificationOptimize protein purification蛋白质晶体学PPT课件 has an even higher purification factor. Virtually no proteins bind to CM columns at pH 8.0.T

40、he use of acidic columns may require passing through the pI of target protein.Hydroxyapatite can give very high purification factors. Size-exclusion chromatography is very useful and normally non-damaging method. Purification by protein properties蛋白质晶体学PPT课件Optimize gene or expressionOptimize gene o

41、r expressionApparent problem MisfoldingLow rate of synthesisProtein degradationExpression systemFusionsor tagsPromoters Expression conditionsCodon biasCo-expressionDomain structurePossible changes 蛋白质晶体学PPT课件MisfoldingMisfoldingFolding efficiencyLack of proper chaperones.Synthesis rateSynthesis is t

42、oo fast for the folding capacity of the system.Protein localizationProtein requires specific compartmentalization (i.e. periplasmic or intramembrane) to fold.Post-translational modificationEukaryotic proteins often require specific PTM to mature. 蛋白质晶体学PPT课件Folding efficiencyToxic proteins are often

43、 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 nutrients or low-level co-factors (e.g. certain metal ions).Protein localizationProt

44、ein 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蛋白质晶体学PPT课件Folding efficiencyIf inclusion bodies are not formed, improperly folded protein can be rapidly

45、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 degradation, e.g.when protein is subjected to signal peptidases in bac

46、terial 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 degradation蛋白质晶体学PPT课件Fusions or tagsCan have a tremendous neg

47、ative 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 definition of domain boundaries can have paramou

48、nt effect on folding. Expression systemFusionsor tagsExpression conditionsFoldingefficiencyCo-expressionDomain structure蛋白质晶体学PPT课件Expression systemIt is easier (and cheaper) to produce massive quantities of proteins in bacteria or yeast.PromotersExpression conditionsTemperature, nutrient/oxygen con

49、tent, 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 systemFusionsor tagsExpression conditionsSynthe

50、sisSynthesisrateratePromotersDomain structureCodon bias蛋白质晶体学PPT课件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 protein stock in small

51、aliquots.Optimize existing sample propertiesOptimize existing sample properties蛋白质晶体学PPT课件II. Protein CrystallizationII. Protein Crystallization蛋白质晶体学PPT课件General approach for protein crystallizationMacromolecular crystals are composed of approximately 50% solvent on average, though this may vary fr

52、om 25 to 90% depending on the particular macromolecule.Macromolecular 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 screeningCrystal optimizationTwo steps for prote

53、in crystal obtaining蛋白质晶体学PPT课件蛋白质晶体学PPT课件ScreeningRoboticManualCheap Time-tested Readily availableAllows for creativityMultitude of conditions Highly reproducible Easy to document and track data Lower consumption of protein蛋白质晶体学PPT课件蛋白质晶体学PPT课件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;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 inter