蛋白质晶体学-ppt课件.ppt

1、Protein Protein Crystallography Crystallography 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射线并因此获得射线并因此获得1901年年首届诺贝尔物理学奖，首届诺贝尔物理学奖，X射线历经射线历经110年跨越年跨越3个世纪，由于个世纪，由于众多学者在探索众多学者在探索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位物理学家、晶体学家、化学家、分子生物学家等位物理学家、晶体学家、化学家、分子生物学家等分别获得了物理（分别获得了物理（7项）、化学（

2、项）、化学（9项）、生理学或医学（项）、生理学或医学（3项）项）总计总计19项诺贝尔奖。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

3、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。 并最终使并最终使X X射线衍射成为有机分子（特别是生物活性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、结构功能关物分

4、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。系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年代中期年代中期，贝纳尔和藿，贝纳尔和藿奇金开始用奇金开始用X X射线衍射方法研究胃蛋白酶的晶体结构，但直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，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衍射实验数据建立了脱氧核衍射实验数据建立了脱氧核糖核酸

5、糖核酸(DNA)(DNA)的双螺旋结构，并因此获得的双螺旋结构，并因此获得19621962年的诺贝尔生年的诺贝尔生理学和医学奖。理学和医学奖。 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衍射方法研究衍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，历经的晶体结构，历经20多年的艰苦努力，多年的艰苦努力，在众多科学家的共同参与下，终于在在众多科学家的共同参与下，终于在1960年获得了这两个蛋年获得了这两个蛋白质的三维结构，并因此荣获白质的三维结构，并因此荣获1962年的诺贝尔化学奖。年的诺贝尔化学奖。 在在1957至至1967年的年的10年中，相继用年中，相继用

6、X衍射方法测定了衍射方法测定了溶菌酶溶菌酶、胰岛素胰岛素、胰凝乳蛋白酶胰凝乳蛋白酶A、核糖核酸酶核糖核酸酶、核糖核酸核糖核酸酶酶S和和羧肽酶羧肽酶的高分辨晶体结构。的高分辨晶体结构。 戴森豪菲尔和胡贝尔、米海尔因测定戴森豪菲尔和胡贝尔、米海尔因测定紫色细菌光合作用紫色细菌光合作用中心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，形成了新的蛋白质晶体学科蛋白质晶体学科与与结构分子生物学科结构分子生物学科。 物理奖 （7项 8人） 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1914M

7、ax 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.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

8、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 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)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

9、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 Hodgkin(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

10、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 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

11、 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. Hounsfield(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2 X射线晶体结构分析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

12、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/H269DLicT wt (inactive)Comparison of licT-wt and licT mutantGraille* and Zhou* et al. 2004 van Tilbeurgh et al. EMBO J. 2001Yang et al. EMBO J. 20021122mRNA1122mRNAPKD=10M KD=1MCATPRD2PRD1RATCATRNA Structure-dir

13、ected drug design An example of Thy1 from Thermotoga maritima Thy1: thymidylate synthase-complementing protein present in archaea, prokaryotes, viruses NOT in eukaryotesLesley, SA et al. PNAS; 2002Thy1-FAD-dUMP Thy1-dUMP-HEPES PDB Content Growth (2004/08/01)52,66227,99916,0975,816 5,6992,133 1,03189

14、2645010,00020,00030,00040,00050,00060,000targetsclonedexpressedsolublepurifiedcrystallizeddiffraction-qual.diffractionstructuresOutput from International Structural Genomics ConsortiaContribution from crystallographers, 2004/04/132,75583824920005001,0001,5002,0002,5003,000targetsHSQCNMR assignedNMR

15、structuresOutput from International Structural Genomics Consortiium Contribution from NMR spectrometrists, 2004/04/13Future orientations of SG1, Reconstruction of multiprotein complexes (based on interactomics)2, Systematically solving the 3-D structures of membrane proteins (a challenge of novel te

16、chniques)3, Systems Biology Interactomes:1, Yeast two-hybrid2, TAP (tandem affinity purification)3, Mass Spectrometry4, Co-IP (coimmunoprecipitation)5, Phage displayOverexpress the putative protein complex in vivoor Reconstruct it in vitro from the individual proteins Solve the 3-D structure by mean

17、s of X-ray crystallography Cryo-Electron Microscopy Electron crystallography (2D EM) Electron tomographySystematically Structure the Membrane Proteins: A big challenge!PDB: 26,880 structures, updated on 2004/08/24http:/www.rcsb.org/pdb/index.html Membrane proteins: 81 structures, updated on 2004/06/

18、15 http:/www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.htmlStructural Biology ProcessesStructural 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 interestIdeal caseIdeal caseTragic realityTragic realityDesign multipleconstructsStudy literature and analog/model casesEvaluate and

19、optimize expressionSmall-scale purificationEvaluate proteinqualityLarge-scale purificationScreeningSelect expression system(s)Only a few (or one)constructsNew protein withlittle prior knowledgeSub-optimal expressionPurificationLimited choice ofexpression systemsI. Recombinant protein over-I. Recombi

20、nant protein over-expression and purificationexpression and purificationExpression systems:1. Bacteria system2. Yeast3. Insect cells4. Mammalian cells5. Cell-free systemSome Vectors for E.coli Expression SystemProtein Expression in YeastCloning of target gene to vectorTransform to yeast Pichia pasto

21、risSelection of recombinant yeast strainYeast cell culture for protein productionProtein Expression in Insect CellsAfter recombinationCloning of target gene to pFastBacTransform to bacteria with BacmidBacmid transfected to insect cellsVirus assembly in insect cellsViruses infect Insect Cells for pro

22、tein productionStrains for expression: Sf9, Sf21, Hi5Transient Expression In Mammalian Cells293E cell can be cultured in suspension medium Recombinant plasmid with target geneTransfect to 293E cells with PEIHarvest cells for protein purification293EBNA1 Cells With GFP Expressing VectorABA. Whole cel

23、ls on plate; B. Cells in the same plate to A viewed by GFP florescenceRecombinant Proteins Expression In 293EBNA1 Cells Lanes: 1. Protein standard; 2. Control whole 293E cells; 3. GFP expressed 293E cells; 4. HCF-1N380 expressed 293E cells; 5. HCF-1N16-363 expressed 293E cells. Recombinant protein 1

24、 (lane 4)1 2 3 4 5142031456794Recombinant protein 2 (lane 5)GFP (lane3)Cell-free System for Protein ProductionSometimes it can produce soluble protein which can not be expressed as soluble form with cellular system.Roche: Rapid Translation System (RTS) Rapid protein expressionToxic protein expressio

25、n1. ProteinProtein Complex Expression and Purification: a. Proteins express separately; b. Proteins co-express in one cell.2. Protein-Nucleic Acid： a. Protein-DNA Complex； b. Protein-RNA Complex.Producing Protein Complexes Producing Protein Complexes for Crystallizationfor CrystallizationMethods for

26、 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-polycistronic；Benefits of in vivo reconstitution (coexpression)efficiency one round of

27、 expression one round of purificationquality coexpression and cofolding of polypeptides in the presence of cellular chaperones may increase yield of functional complexProteinProtein Complex Expression and PurificationProteinProteinDNADNA ComplexComplex1. Protein solubility: higher in high salt buffe

28、r usually;2. Protein-DNA complex stability: more stable than protein alone;3. 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 reaction intermediate: disulfide bridge; prote

29、in point mutation, etc;6. Preparation of protein-DNA complexes: mix with extra molar DNA;7. Crystallization: PEG or MPD in low slat buffer;8. Example: over 6000 trial for protein-DNA complex.Protein-RNA ComplexDifficulties: avoid of RNase! 1. Phosphate groups interfere crystal packing; 2. Elongated

30、RNAs pack loosely;RNA engineering: blunt or sticky ends; deletion, replacement, etc;RNA preparation: 1. Synthesis; 2. In vitro transcription;Protein Modification for Crystallization1. Protein inhibitor, partner and monoclonal antibody;2. Protein post-translational modification;3. Protein mutagenesis

31、: truncation, mutation, deletionProtein Mutagenesis1. Truncation or deletion: secondary structure prediction; DXMS result; homologue protein sequences comparison or structure comparison;2. Mutation methods a. Selected point mutation; b. Random mutation：DNA shuffling for chimeric protein; random muta

32、tion by low-fidelity PCR.Hydrogen/deuterium exchange mass spectroscopyHydrogen/deuterium exchange mass spectroscopy (DXMS) for protein analysis(DXMS) for protein analysis Keenan, Robert J. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 8887-8892Random mutation by DNA shufflingRandom mutation by DNA s

33、huffling Mutation selection by GFP folding reporter GFPTarget ProteinCorrect Folding of Target ProteinMisfolding of Target ProteinFluroscenceNH3+COO-No Fluroscence(Waldo, GS. Et al.1999,Nature Biotech.17:691)GFP Folding ReporterGFP Folding Reporter GFPTargetpGFPuvWild-type geneRandom mutagenesis wit

34、h Polymerase(Exo-)-Random Mutagenesis.-Clone into GFP vector.-Select the brightest colonies.-Test the solubility of Kelch-GFP.-Reclone into GST fusion vector.-Test the solubility of GST-Kelch.PCRProtein purification methodProtein purification method1. Affinity Column: by tags or antibodies;2. Ion ex

35、change column;3. Size exclusion column;4. Hydrophobic interaction;5. othersMetal 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 sinc

36、e 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 elute at moderate ionic strength. DNA precipitation (e.g. via polyethyleneimine addition) is a useful, but somewhat ris

37、ky 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 protein is bound. A CM-columnOptimize protein purificationOptimize protein purification has an even higher purifi

38、cation 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 chromatography is very useful and normally non-damaging method. Purification by prot

39、ein propertiesOptimize gene or expressionOptimize gene or expressionApparent problem MisfoldingLow rate of synthesisProtein degradationExpression systemFusionsor tagsPromoters Expression conditionsCodon biasCo-expressionDomain structurePossible changes MisfoldingMisfoldingFolding efficiencyLack of p

40、roper 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 proteins often require specific PTM to mature. Folding

41、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 nutrients or low-level co-factors (e.g. certain me

42、tal 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 synthesisFolding efficiencyIf inclusion bodies are not formed, improperly folded

43、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 degradation, e.g.when protein is subjected to s

44、ignal 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 degradationFusions or tagsCan have a tr

45、emendous 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 definition of domain boundaries can

46、have paramount effect on folding. Expression systemFusionsor tagsExpression conditionsFoldingefficiencyCo-expressionDomain structureExpression systemIt is easier (and cheaper) to produce massive quantities of proteins in bacteria or yeast.PromotersExpression conditionsTemperature, nutrient/oxygen co

47、ntent, 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 conditionsSynth

48、esisSynthesisrateratePromotersDomain structureCodon biasAvoid 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 aliquots.O

49、ptimize existing sample propertiesOptimize existing sample propertiesII. Protein CrystallizationII. Protein CrystallizationGeneral approach for protein crystallizationMacromolecular crystals are composed of approximately 50% solvent on average, though this may vary from 25 to 90% depending on the pa

50、rticular 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 protein crystal obtainingScreeningRob