蛋白質晶體學2010

1、Protein Protein Crystallography Crystallography 一、概述一、概述 1 1 歷史的回顧歷史的回顧 1895年德國物理學家倫琴發(fā)現年德國物理學家倫琴發(fā)現X射線并因射線并因此獲得此獲得1901年首屆諾貝爾物理學獎，年首屆諾貝爾物理學獎，X射線歷射線歷經經110年跨越年跨越3個世紀，由于眾多學者在探索個世紀，由于眾多學者在探索X射線性質、應用、儀器等方面的創(chuàng)新性研究，射線性質、應用、儀器等方面的創(chuàng)新性研究，先后有先后有29位物理學家、晶體學家、化學家、分位物理學家、晶體學家、化學家、分子生物學家等分別獲得了物理（子生物學家等分別獲得了物理（7項）、化學項

2、）、化學（9項）、生理學或醫(yī)學（項）、生理學或醫(yī)學（3項）總計項）總計19項諾貝項諾貝爾獎。爾獎。 1912年勞厄獲得了X射線通過晶體后產生的衍射斑點圖像（勞厄衍射圖），證明了X射線的波動性及其波長范圍。隨后提出了表示原子排列周期與X射線波長間關系的著名的衍射方程（勞厄方程），并成功地解釋了晶體衍射的實驗結果。 英國物理學家布拉格父子、達爾文等人發(fā)展了X射線衍射理論，類比光學反射原理提出了表示晶體結構（晶面間距d）、X射線波長（）與衍射方位（）間的關系的布拉格方程，提出了嵌鑲晶體、完整晶體和包含有原子熱運動諸因素的衍射強度公式，闡明了X射線通過晶體產生衍射的付里葉變換本質，獲得了X射線的連續(xù)光

3、譜與取決于陰極材料的特征光譜。 康普頓發(fā)現了X射線二次散射時引發(fā)的波長的變化(康普頓-吳有訓散射)而確定了其粒子性質，從而揭示了X射線的波動與粒子二象性。 之后，全世界眾多的物理實驗室相繼開展了對X射線的基礎研究工作，并逐步拓展為一個多學科交叉研究熱點，主要的應用領域包括：礦物學、物理學、有機與無機化學、分子生物學、醫(yī)藥學、金屬與材料科學等。 并最終使并最終使X X射線衍射成為有機分子（特別射線衍射成為有機分子（特別是生物活性分子）立體結構測定的有力工具，是生物活性分子）立體結構測定的有力工具，為研究生理活性物質（藥物分子）的立體結為研究生理活性物質（藥物分子）的立體結構、結構改造、結構預測、

4、結構功能關系構、結構改造、結構預測、結構功能關系為目標的有機晶體學科奠定了基礎。為目標的有機晶體學科奠定了基礎。 對于對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，貝納爾和藿奇金開始用，貝納爾和藿奇金開始用X X射線衍射方法研射線衍射方法研究胃蛋白酶的晶體結構，但直到布拉格主持究胃蛋白酶的晶體結構，但直到布拉格主持凱文迪實驗室后，才使得這一工作取得突破，凱文迪實驗室后，才使得這一工作取得突破，為創(chuàng)建為創(chuàng)建分子生物學科分子生物學科奠定了基礎。奠定了基礎。 1953 1953年沃森和克里克根據年沃森和克里克根據X X衍射實驗數據衍射實驗數據建立了脫氧核糖核酸建立了脫氧核糖

5、核酸( (DNA)DNA)的雙螺旋結構，并的雙螺旋結構，并因此獲得因此獲得19621962年的諾貝爾生理學和醫(yī)學獎。年的諾貝爾生理學和醫(yī)學獎。 肯德魯和佩盧茨從肯德魯和佩盧茨從30年代開始，應用年代開始，應用X衍衍射方法研究射方法研究肌紅蛋白肌紅蛋白與與血紅蛋白血紅蛋白的晶體結構，的晶體結構，歷經歷經20多年的艱苦努力，在眾多科學家的共多年的艱苦努力，在眾多科學家的共同參與下，終于在同參與下，終于在1960年獲得了這兩個蛋白年獲得了這兩個蛋白質的三維結構，并因此榮獲質的三維結構，并因此榮獲1962年的諾貝爾年的諾貝爾化學獎?；瘜W獎。 在在1957至至1967年的年的10年中，相繼用年中，相繼用

6、X衍衍射方法測定了射方法測定了溶菌酶溶菌酶、胰島素胰島素、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體結構。高分辨晶體結構。 戴森豪菲爾和胡貝爾、米海爾因測定戴森豪菲爾和胡貝爾、米海爾因測定紫紫色細菌光合作用中心色細菌光合作用中心的三維結構而獲得的三維結構而獲得1988年的諾貝爾化學獎，形成了新的年的諾貝爾化學獎，形成了新的蛋白質晶體蛋白質晶體學科學科與與結構分子生物學科結構分子生物學科。 物理獎 （7項 8人） 成成 就就(另一發(fā)現者Mosley因第一次世界大戰(zhàn)陣亡而未獲獎)年代獲獎者成 就1901Wilhelm Konrad Rnt

7、gen (德)W.K.倫琴1895年發(fā)現X射線及其性質1914Max Von Laue(德) M.J.勞厄1912年發(fā)現晶體X射線衍射1915William Henry Bragg(英)W.H.布拉格1912年建立X射線衍射晶體結構分析William Lawrence Bragg(英)W.L.布拉格1917Charles Glouer Barkla(英)C.G.巴克拉1909年建立X射線光譜學的K、L系(另一發(fā)現者Mosley因第一次世界大戰(zhàn)陣亡而未獲獎)1924Karl Manne Georg Siegbahn(瑞典)K.M.G.西格班1912年發(fā)現X射線光譜學的M系1927Arthur H

8、olly Compton(美)A.H.康普頓1919年發(fā)現X射線能量變化的康普頓效應1981Kai M. Siegbahn(瑞典)K.西格班1956年發(fā)現X射線光電子能譜 化學獎 （9項 15人）1936Peter Debye(荷）P.德拜1916年 提出粉末X射線晶體結構分析1946James Batchelle Sumner(美)J.B.薩姆納1926年獲得尿素酶結晶；1937年獲得氧化氫酶結晶等John Noward Northrop(美)J.N.諾思羅普1930年獲得胃蛋白酶結晶等Wendell Stanley(美)W.斯坦利1935年獲得菸草花葉病毒(TMV)結晶；1946年獲得流感

9、病毒結晶1954Linus Pauling(美)L.鮑林用X射線衍射法確定多肽結構的化學鍵本質1962John Cowdery Kendrew(英)J.C.肯德魯用X射線衍射法測定肌紅蛋白結構Max Ferdinand Peruty(英)M.F.佩魯茨用X射線衍射法測定血紅蛋白結構1964Dorothy Mary Cronfoot Hodgkin(英)D.M.C.霍奇金用X射線衍射法測定青霉素與B12分子結構1976William Nunn Lipscomb(美)W.N.利普斯科姆低溫X射線衍射法確定硼氫化合物分子結構1982Aaron Klug(英)A.克盧格蛋白質分子結構的電子顯微鏡三維重

10、組1985Herbert A. Hauptman(美)H.A.豪普特曼X射線衍射分析直接法的建立Jerome Karle(美)J.卡爾1988Hartmut Michel(德)H.米歇爾以X射線衍射法測定了細菌光合作用反應中心的分子立體結構Johann Deisenhifer(德)J.戴森霍菲爾Robert Huber(德)R.胡貝爾 生理醫(yī)學獎 （3項 6人）年代年代獲獎者獲獎者成成 就就1946Hermann Joseph Muller(美)H.J.繆勒發(fā)現X射線照射引起基因突變，建立了輻射遺傳學1962Francis Harry Compton Crick(英)F.H.C.克里克1953

11、年應用X射線衍射法建立了DNA分子結構模型James Dewey Watson(英)J.D.沃森Maurice Hugh Frederick Wilkins(英)M.H.F.威爾金斯1979Allan M. Cormack(美)A.M.科馬克1969年建立了計算機輔助X射線斷層掃描(CT)Godfrey N. Hounsfield(英)G.N.豪斯菲爾德1937Clinton Joseph Davisson(美)C.J.戴維森發(fā)現電子衍射技術1994Clifford Clenwood Shull(美)C.C.沙爾發(fā)現中子衍射技術2 X射線晶體結構分析X射線 ：表示所用的物理源與晶體相互作用的物

12、理效應衍射晶體：表示固體狀態(tài)下的一種特殊存在形態(tài)晶體生長晶體的幾何性質對稱性衍射信息中的對稱性相位計算中的對稱性結構分析：兩次付里葉變換，完成第二次付里葉變換的數學方法晶體結構描述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=1MCATPRD2

13、PRD1RATCATRNA Structure-directed 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,

14、0975,816 5,6992,133 1,031892645010,00020,00030,00040,00050,00060,000targetsclonedexpressedsolublepurifiedcrystallizeddiffraction-qual.diffractionstructuresOutput from International Structural Genomics ConsortiaContribution from crystallographers, 2004/04/132,75583824920005001,0001,5002,0002,5003,000

15、targetsHSQCNMR assignedNMR 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 protei

16、ns (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 displayOverexpress the putative protein complex in vivoor Reconstruct it in vitro from the individual proteins Solv

17、e the 3-D structure by means 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 2004/08/24/pdb/index.html Membrane proteins: 81 stru

18、ctures, updated on 2004/06/15 http:/www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.htmlStructural Biology ProcessesStructural Biology Processes X射線衍射實驗和結構計算過程Fourier變換與Fourier反變換Gene of interestIdeal caseIdeal caseTragic realityTragic realityDesign multipleconstructsStudy literature and anal

19、og/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 ofexpression systemsI. Recombin

20、ant protein over-I. Recombinant protein over-expression and purificationexpression and purificationExpression systems: Bacteria system Yeast Insect cells Mammalian cells1. Cell-free systemSome Vectors for E.coli Expression SystemProtein Expression in YeastCloning of target gene to vectorTransform to

21、 yeast Pichia pastorisSelection 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 I

22、nsect Cells for protein 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 V

23、ectorABWhole cells on plate; 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. Recombin

24、ant protein 1 (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 prot

25、ein expressionProteinProtein 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 CrystallizationM

26、ethods 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-polycistronic；Benefits of in vivo reconstitution (coexpression)efficiency on

27、e round of 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 ComplexComplexProtein solubility: higher in high sal

28、t 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 reaction intermediate: disulfide bridge; prot

29、ein 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 ComplexDifficulties: avoid of RNase! 1. Phosphate groups interfere crystal packing; 2. Elongated RNAs

30、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: tru

31、ncation, mutation, deletionProtein Mutagenesis1. 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 mutation by

32、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 Random mutation by DNA shufflingshuffling

33、 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 with Polyme

34、rase(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 methodAffinity Column: by tags or antibodies;Ion exchange column;

35、Size exclusion column;Hydrophobic interaction;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 since its purification fact

36、or 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 risky step.Most proteins d

37、o 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 purification factor. Virtuall

38、y 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 protein propertiesOptimize

39、gene or expressionOptimize gene or expressionApparent problem MisfoldingLow rate of synthesisProtein degradationExpression systemFusionsor tagsPromoters Expression conditionsCodon biasCo-expressionDomain structurePossible changes MisfoldingMisfoldingFolding efficiencyLack of proper chaperones.Synthe

40、sis 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 efficiencyToxic protein

41、s 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 metal ions).Protein local

42、izationProtein 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 protein can be rapidly

43、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

44、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 degradationFusions or tagsCan have a tremendous negative or po

45、sitive 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 paramount effect o

46、n 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 content, antibiotics, etc

47、. 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 conditionsSynthesisSynthesisrateratePr

48、omotersDomain 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.Optimize existing sample

49、 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 particular macromolecule.

50、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 obtainingScreeningRoboticManualCheap Time-te

51、sted Readily availableAllows for creativityMultitude of conditions Highly reproducible Easy to document and track data Lower consumption of protein1. 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. Alt