蛋白質(zhì)分子設(shè)計(jì)課件2

蛋白質(zhì)分子設(shè)計(jì)蛋白質(zhì)分子設(shè)計(jì)蛋白質(zhì)分子設(shè)計(jì)是一門新興的研究領(lǐng)域，其本身在不斷地發(fā)展，其內(nèi)容也在不斷地更新。蛋白質(zhì)分子設(shè)計(jì)方面的主要內(nèi)容與研究進(jìn)展，以及應(yīng)用分子設(shè)計(jì)取得的成功實(shí)例。蛋白質(zhì)分子的特殊性蛋白質(zhì)分子功能的重要性蛋白質(zhì)分子應(yīng)用的局限性蛋白質(zhì)分子結(jié)構(gòu)的脆弱性蛋白質(zhì)工程與蛋白質(zhì)設(shè)計(jì)蛋白質(zhì)設(shè)計(jì)及結(jié)構(gòu)與功能關(guān)系研究的必要性;蛋白質(zhì)設(shè)計(jì)是多學(xué)科的交叉領(lǐng)域;蛋白質(zhì)設(shè)計(jì)在許多方面取得的顯著進(jìn)展Proteinengineering:bywhichwemeanmutatingthegeneofanexistingproteininanattempttoalteritsfunctioninapredictablewayProteinDesign:whichhasthemoreambitiousgoalofdesigningdenovoaproteintofulfilladesiredfunction第一節(jié)基于天然蛋白質(zhì)結(jié)構(gòu)的分子設(shè)計(jì)

一、概述蛋白質(zhì)結(jié)構(gòu)與功能的關(guān)系的認(rèn)識對蛋白質(zhì)設(shè)計(jì)是至關(guān)重要的，蛋白質(zhì)的結(jié)構(gòu)涉及一級結(jié)構(gòu)(序列)及三維結(jié)構(gòu)。即使蛋白質(zhì)的三維結(jié)構(gòu)是已知的，選擇一個合適的突變體仍是困難的，這說明蛋白質(zhì)設(shè)計(jì)任務(wù)的艱巨性，它涉及多種學(xué)科的配合，如計(jì)算機(jī)模擬專家、X射線晶體學(xué)家、蛋白質(zhì)化學(xué)家、生物技術(shù)專家等的合作與配合。

計(jì)算機(jī)模擬

基因構(gòu)建突變蛋白質(zhì)產(chǎn)品功能分析蛋白質(zhì)設(shè)計(jì)循環(huán)蛋白質(zhì)分子設(shè)計(jì)大致涉及的幾個重要方面在蛋白質(zhì)設(shè)計(jì)開始之前，要對所要求的活性進(jìn)行篩選。由于真菌與細(xì)胞相對容易處理，因此它們是一個生物活性物質(zhì)源。由于基因工程的發(fā)展，真核基因表達(dá)技術(shù)的發(fā)展使動物蛋白質(zhì)與植物蛋白質(zhì)的數(shù)目迅速增長，又增加了新的生物活性物質(zhì)源。

蛋白質(zhì)分子設(shè)計(jì)大致涉及的幾個重要方面

篩選以及純化蛋白質(zhì)需要進(jìn)行細(xì)致的表征，測定它們的序列、三維結(jié)構(gòu)、穩(wěn)定性、催化活性等。專一性突變產(chǎn)物是蛋白質(zhì)設(shè)計(jì)成敗的關(guān)鍵。一些新技術(shù)，如PCR及自動化技術(shù)的發(fā)展使各種類型的基因工程變得快速、容易。計(jì)算機(jī)模擬技術(shù)在蛋白質(zhì)設(shè)計(jì)循環(huán)中占有重要位置。建立蛋白質(zhì)三維結(jié)構(gòu)模型，確立突變位點(diǎn)或區(qū)域以及預(yù)測突變后的蛋白質(zhì)的結(jié)構(gòu)與功能對蛋白質(zhì)工程是至關(guān)重要的。在明確突變位點(diǎn)或蛋白質(zhì)序列應(yīng)改變的區(qū)域后，可以進(jìn)行定位突變，但要得到具有預(yù)期結(jié)構(gòu)與功能的蛋白質(zhì)是不容易的，可能需要經(jīng)過幾輪的循環(huán)。蛋白質(zhì)三維結(jié)構(gòu)知識的必要性

蛋白質(zhì)三維結(jié)構(gòu)知識對于蛋白質(zhì)工程是絕對必要的。目前PDB(ProteinDataBank)已收集數(shù)以萬計(jì)個蛋白質(zhì)晶體結(jié)構(gòu)，但是通常蛋白質(zhì)序列的數(shù)目比蛋白質(zhì)三維結(jié)構(gòu)的數(shù)目大100倍。當(dāng)我們開始對某一天然蛋白質(zhì)進(jìn)行蛋白質(zhì)分子設(shè)計(jì)時，首先要查找PDB了解這個蛋白質(zhì)的三維結(jié)構(gòu)是否已被收錄。如果PDB中沒有收錄又未見文獻(xiàn)報道，我們需要通過蛋白質(zhì)X射線晶體學(xué)及NMR方法測定蛋白質(zhì)的三維結(jié)構(gòu)，或者通過結(jié)構(gòu)預(yù)測的方法構(gòu)建該蛋白質(zhì)三維結(jié)構(gòu)模型。設(shè)計(jì)目標(biāo)及解決辦法

蛋白質(zhì)結(jié)構(gòu)與功能的關(guān)系對于蛋白質(zhì)工程及蛋白質(zhì)分子設(shè)計(jì)都是至關(guān)重要的。如果我們想改變蛋白質(zhì)的性質(zhì)，必須改變蛋白質(zhì)的序列。Hartley等于1986年完成了一個我們所要的有關(guān)蛋白質(zhì)重要性質(zhì)設(shè)計(jì)目標(biāo)及解決辦法表，該表至今仍有參考價值。

設(shè)計(jì)目標(biāo)

解決辦法熱穩(wěn)定性對氧化的穩(wěn)定性對重金屬的穩(wěn)定性pH穩(wěn)定性提高酶學(xué)性質(zhì)引入二硫橋增加內(nèi)氫鍵數(shù)目改善內(nèi)疏水堆積增加表面鹽橋把Cys轉(zhuǎn)換為Ala或Ser把Met轉(zhuǎn)換為Gln、Val、Ile或Leu把Trp轉(zhuǎn)換為Phe或Tyr把Cys轉(zhuǎn)換為Ala或Ser把Met轉(zhuǎn)換為Gln、Val、Ile或Leu替代表面羧基替換表面荷電基團(tuán)His、Cys以及Tyr的置換內(nèi)離子對的置換專一性的改變增加逆轉(zhuǎn)數(shù)(turnovernumber)改變酸堿度蛋白質(zhì)設(shè)計(jì)的目標(biāo)及解決辦法二、蛋白質(zhì)設(shè)計(jì)原理

①內(nèi)核假設(shè)。所謂內(nèi)核是指蛋白質(zhì)在進(jìn)化中保守的內(nèi)部區(qū)域。在大多數(shù)情況，內(nèi)核由氫鍵連接的二級結(jié)構(gòu)單元組成。②所有蛋白質(zhì)內(nèi)部都是密堆積(很少有空穴大到可以結(jié)合一個水分子或惰性氣體)，并且沒有重疊。③所有內(nèi)部的氫鍵都是最大滿足的(主鏈及側(cè)鏈)。④疏水及親水基團(tuán)需要合理地分布在溶劑可及表面及不可及表面。⑤在金屬蛋白中，配位殘基的替換要滿足金屬配位幾何。⑥對于金屬蛋白，圍繞金屬中心的第二殼層中的相互作用是重要的。⑦最優(yōu)的氨基酸側(cè)鏈幾何排列。⑧結(jié)構(gòu)及功能的專一性。形成獨(dú)特的結(jié)構(gòu)，獨(dú)特的分子間相互作用是生物相互作用及反應(yīng)的標(biāo)志。實(shí)踐表明這是蛋白質(zhì)設(shè)計(jì)最困難的問題。三、蛋白質(zhì)設(shè)計(jì)中的結(jié)構(gòu)－功能關(guān)系研究蛋白質(zhì)結(jié)構(gòu)－功能關(guān)系的重要性定位突變在蛋白質(zhì)結(jié)構(gòu)與功能關(guān)系研究中的作用

1.根據(jù)結(jié)構(gòu)信息確定殘基的突變：酪氨酰t-RNA合成酶

2.其他實(shí)驗(yàn)方法鑒定功能殘基：HIV-1蛋白酶；GM-CSF；

3.利用蛋白質(zhì)同源性鑒定功能殘基：保守殘基構(gòu)象的探測定位突變種類插入一個或多個氨基酸殘基；刪除一個或多個氨基酸殘基；替換或取代一個或多個氨基酸殘基。最大量的定位突變是在體外利用重組DNA技術(shù)或PCR方法。突變的加和性原則突變蛋白質(zhì)結(jié)構(gòu)的評估溶解性熱力學(xué)分析Ｘ射線晶體學(xué)及ＮＭＲ譜園二色散方法單克隆抗體探測構(gòu)象變化結(jié)構(gòu)與功能的容忍度

蛋白質(zhì)結(jié)構(gòu)及功能對殘基的替換有一定的容忍度，即結(jié)構(gòu)與功能關(guān)系有一定的穩(wěn)健度。Fersht等替換了Barnase的所有內(nèi)核殘基。結(jié)果表明23％的突變體保留了酶的活性。Mathews及其合作者在溶菌酶內(nèi)核中替換多至10個殘基。實(shí)驗(yàn)證明多重取代的蛋白仍具有活性以及協(xié)同折疊。這些結(jié)果說明不同的氨基序列具有相近的設(shè)計(jì)的結(jié)構(gòu)。四.天然蛋白質(zhì)的剪裁分子剪裁：替換蛋白質(zhì)分子的一個肽段，或一個結(jié)構(gòu)域1）抗體工程（ChapterVI）2）Rop

第二節(jié)全新蛋白質(zhì)設(shè)計(jì)特征：全新蛋白質(zhì)設(shè)計(jì)是另一類蛋白質(zhì)工程，合成具有特異結(jié)構(gòu)與功能的新蛋白質(zhì)。根據(jù)所希望的結(jié)構(gòu)及功能設(shè)計(jì)蛋白質(zhì)或多肽的氨基酸序列。蛋白質(zhì)的全新設(shè)計(jì)內(nèi)容蛋白質(zhì)結(jié)構(gòu)的從頭設(shè)計(jì)蛋白質(zhì)功能的從頭設(shè)計(jì)取得的進(jìn)展：血紅素結(jié)合蛋白、氧化還原活性蛋白質(zhì)、DNA結(jié)合蛋白及基于蛋白質(zhì)的高分子材料。一、蛋白質(zhì)結(jié)構(gòu)的從頭設(shè)計(jì)二級結(jié)構(gòu)模塊單元的自組裝配體誘導(dǎo)組裝通過共價交叉連接實(shí)現(xiàn)肽的自組裝：-S-S-；DAB在合成模板上的肽自組裝線性多肽折疊為球狀結(jié)構(gòu)基于組合庫的全新蛋白質(zhì)設(shè)計(jì)

二、蛋白質(zhì)功能的全新設(shè)計(jì)

蛋白質(zhì)設(shè)計(jì)的目標(biāo)是產(chǎn)生既能折疊為預(yù)想的結(jié)構(gòu)又具有有趣和有用的功能。功能設(shè)計(jì)主要涉及鍵合及催化。為達(dá)到這些目的可以采用兩條不同的途徑：反向?qū)崿F(xiàn)蛋白質(zhì)與工程底物的契合，改變功能；從頭設(shè)計(jì)功能蛋白質(zhì)。蛋白質(zhì)的功能設(shè)計(jì)１．通過反向擬合天然蛋白質(zhì)設(shè)計(jì)新的功能２．鍵合及催化的從頭設(shè)計(jì)３．在全新蛋白質(zhì)中引入結(jié)合位點(diǎn)４．催化活性蛋白質(zhì)的設(shè)計(jì)５．膜蛋白及離子通道的設(shè)計(jì)６．新材料的設(shè)計(jì)

第三節(jié)計(jì)算蛋白質(zhì)設(shè)計(jì)蛋白質(zhì)設(shè)計(jì)是一個理論與實(shí)驗(yàn)之間的循環(huán)。這個循環(huán)已經(jīng)在蛋白質(zhì)的合理設(shè)計(jì)中得到了許多重要進(jìn)展。計(jì)算蛋白質(zhì)設(shè)計(jì)包括能量表達(dá)、能量優(yōu)化、側(cè)鏈構(gòu)象的離散化、殘基分類(內(nèi)核、表面、邊界)、功能位點(diǎn)設(shè)計(jì)、專一性、穩(wěn)定性及序列空間的穩(wěn)健性預(yù)測等方面內(nèi)容。二、蛋白質(zhì)的功能設(shè)計(jì)1）通過反向Mimicking天然蛋白質(zhì)設(shè)計(jì)新功能2）鍵合及催化的從頭設(shè)計(jì)3）在全新蛋白質(zhì)中引入結(jié)合位點(diǎn)4）催化活性蛋白質(zhì)的設(shè)計(jì)5）膜蛋白及離子通道的設(shè)計(jì)6）新材料的設(shè)計(jì)IntroductiontoStructuralBiology:

prediction,engineering,anddesignofproteinstructuresProteinscanbemademorestablebyengineeringThefactorsthatareimportanttoproteinstabilitycanberevealedbydoingproteinengineeringstudies.Anexample:T4lysozyme(fromtheworkdonebyBrianMathews,Univ.ofOregon).T4lysozyme(a)Isa164-aapolypeptidechainthatfoldsintotwodomains:TheN-terminaldomainisof+type,andtheC-terminaldomaincomprises7shorthelices.(b)Hasnodisulfidebonds(c)HastwoCysresidues,Cys54andCys97(thatarefarapartinthefoldedstructure)T4lysozyme(contd.)Tm(themeltingtemperature)Tm(themeltingtemperature):thetemperatureatwhich50%oftheenzymeisinactivated(ormorerigorously,50%oftheenzymeisunfolded)duringreversibleheatdenaturation.ThehigherTm,themorestabletheprotein.WTT4lysozyme’sTm:41.9°CThermodynamicparametersobtainedfromDSCmeasurementsDSC:DifferentialScanningCalorimetry差示掃描量熱法DeterminationofHandSfromDSCCpTThermaldenaturationofaproteinThreemethodstoengineeramorethermostableproteinthanwildtypeT4lysozyme(1)reducingthedifferenceinentropybetweenfoldedandunfoldedprotein.(inpractice,reducingthenumberofconformationsintheunfoldedstate)(2)stabilizingthe

helices.(3)increasingthenumberofhydrophobicinteractionsintheinteriorcore.DisulfidebridgesincreaseproteinstabilityOnewaytoreducethenumberofunfoldedconformationsistointroduceadisulfidebridge.ThegeometryoftheCH2-S-S-CH2-bridgeinproteinsisconfinedtorathernarrowconformationallimits.Thus,deviationsfromthisgeometrywillintroducestrainsintothefoldedstructure.So,pairsofidealpositionsneededtobesoughtinordertoaccommodatedisulfidebridges.Disulfidebridgesincreaseproteinstability(contd.)Threecandidatedisulfidebridgesremainedafterthislongfilteringprocess:3-97,9-164,21-142(bytheway,Cys54wasmutatedtoThrtoavoidtheformationofincorrectdisulfidebondsduringfolding).Disulfidebridgesincreaseproteinstability(contd.)4.8°c6.4°c11°COxidizedmutantsaremorestablethanWT.(b)ReducedmutantsarelessstablethanWT.(c)Thelongertheloopbetweenthecysteineresiduesofthemutantswithsingledisulfidebonds,thelargerwastheeffectonstability.(d)Themutationaleffectswereadditive.Triplemutants:4.8+6.4+1122°CDisulfidebridgesincreaseproteinstability(contd.)GlycineandprolinehaveoppositeeffectsonstabilityAglycineresidueataspecificpositioninaproteinhasusuallyoneconformationinafoldedstructurebutcanhavemanydifferentconformationsindifferentunfoldedstructuresofthesameproteinandtherebycontributetothediversityofunfoldedconformations.ProlineresidueshavelessconformationalfreedominunfoldedstructuresthananyotherresidueGlycineandprolinehaveoppositeeffectsonstability(contd.)AnotherwaytodecreasethenumberofpossibleunfoldedstructuresofaproteinistomutateGlyresiduestoanyotherresidueortoincreasethenumberofProresidues.Results:inT4lysozyme,Gly77->Ala(Tmincreaseof1°C)Ala82->Pro(Tmincreaseof2°C)Glycineandprolinehaveoppositeeffectsonstability(contd.)Thethree-dimensionalstructuresofthesemutantenzymeswerealsodetermined:theAla82->Promutanthadastructureessentiallyidenticaltothewildtypeexceptforthesidechainofresidue82;thisstronglyindicatesthattheeffectonTmofAla82->Proisindeedduetoentropychanges.Stabilizingthedipolesof

helicesincreasesstabilityThehelixdipoleconcept:thepositivechargeisattheN-terminusofthehelix,andthenegativechargeisattheC-terminusofthehelix.Thus,negativeionsareusuallyboundtotheN-terminalendofthehelix.Results,inT4lysozyme,Ser38->Asp(Tmincreaseof2°C)Asn144->Asp(Tmincreaseof2°C)Stabilizingthedipolesof

helicesincreasesstability(contd.)Stabilizingthedipolesof

helicesincreasesstability(contd.)MutationsthatfillcavitiesinhydrophobiccoresdonotstabilizeT4lysozyme,althoughtheymaystabilizeotherproteinsTheeliminationofacavityofthesizeofone–CH2-groupintheproteininteriorstabilizestheenzymebyabout1kcal/mol.Results:inT4lysozyme,Leu23->Phe&Ala129->Val:thesenewsidechainswerehydrophobicandlargeenough,inprinciple,tofillthecavitieswithoutmakingtooclosecontactswithsurroundingatoms.MutationsthatfillcavitiesinhydrophobiccoresdonotstabilizeT4lysozyme,althoughtheymaystabilizeotherproteins(contd.)Inpractice,theywerebothlessstablethanwildtypeby0.5to1.0kcal/mol.Itturnsoutthatinordertofillthecavities,thenewsidechainsinthemutantsadoptenergeticallyunfavorableconformations.Thisintroducesstraininthestructure.Mutationsdesignedtofillexistingcavitiesmaybeeffectiveinsomecases,buttheyarenotlikelytoprovideageneralroutetosubstantialimprovementinproteinstability.ProteinscanbeengineeredbycombinatorialmethodsCombinatorialmethods:alsocalledinvitroordirectedevolutiontechniquesCombinatorialmethods:inwhich,librariesofrelatedproteinsareanalyzedsimultaneously.Bysortingtheselibrariestoselectforaparticularfunction,thesmallnumberofactiveproteinscanbeseparatedfrommillionsofinactivevariants.Proteinscanbeengineeredbycombinatorialmethods(contd.)MethodsofgeneratingcombinatorialDNAlibraries:(a)oligonucleotide-directedmutagenesis,(b)error-pronePCR,and(c)DNAshufflingThemutatedgenesarethenselectedinvivobyconferringafunctiontocells,orinvitrobybindingtoimmobilizedtarget.Themostcommonmethodforinvitroselectionisthephagedisplaymethod.MethodsofgeneratingcombinatorialDNAlibrariesMethodsofgeneratingcombinatorialDNAlibraries(contd.)MethodsofgeneratingcombinatorialDNAlibraries(contd.)MethodsofgeneratingcombinatorialDNAlibraries(contd.)Phagedisplaymethod(1)M13(afilamentousphagecontainingss-DNAencasedinaproteincoat):containsfivecoatproteins,twoofwhicharegVIIIp(geneVIIIprotein)andgIIIp(geneIIIprotein).Phagedisplaymethod(2)Phagedisplaymethod(2):(contd.)Phagedisplaymethod(2):(contd.)PhagedisplayofrandompeptidelibrariesidentifiedagonsitsoferythropoietinreceptorAgonists:whichmimicthefunctionofahormonebybindingtoitsreceptorandcausingthenormalresponse.Antagonists:whichbindtothereceptorbutdonotactivatehormone-inducedeffectsPhagedisplayofrandompeptidelibrariesidentifiedagonsitsoferythropoietinreceptor(contd.)Toidentifypeptidesorsmallmoleculeswhicheitherinhibitorstimulatehormonefunction.Thehot-spotprinciple:asmallnumberofresiduesatabindinginterfacecontributemostofthebindingenergy.Erythropoietin(EPO):acytokinehormonewhichstimulatesformationofredbloodcells.EPOR:erythropoietinreceptorEBP:extra-cellulardomainofEPORPhagedisplayofrandompeptidelibrariesidentifiedagonsitsoferythropoietinreceptor(contd.)NicholasWrightonandcoworkersusedthephagedisplaymethodstoisolatea20residuepeptide,calledEMP1,whichmimicstheactivityofEPObypromotingdimerizationandactivationofEPOR.EMPIwasselectedbytwocyclesofphagedisplay.Phagedisplayofrandompeptidelibrariesidentifiedagonsitsoferythropoietinreceptor(contd.)First,randompeptidelibrariesofthesequenceCX8CweredisplayedasgVIIIpfusions.MultivalentgVIIIproteinfusiondisplaypermittedselectionofweakbindersbyavidity(multiple)bindingtoimmobilizeddimersofEBP.TheseweaklybindingpeptideswereexpandedtotheformofX5CX8CX3andparticularlyrandomizedintheX8residues.FusionofthisnewlibrarytogIIIpyieldedalowervalencyofdisplayandallowedselectionoftightbinders.EMP1,isolatedfromthislibrary,boundtoEBPwithaKdof0.2

MandstimulatedEPORactivityinvivoEachpeptidemonomerformsahairpin,stabilizedbyanintramoleculardisulfidebondDNAshufflingallowsacceleratedevolutionofgenesDNAshufflingallowsacceleratedevolutionofgenes(contd.)DNAshufflingallowsacceleratedevolutionofgenes(contd.)DNAshufflingallowsacceleratedevolutionofgenes(contd.)Thedifferentcolorsidentifytheoriginofeachproteinsegment;Interestingly,thesesegmentsformunitsofsecondarystructure.AmodelbasedontheX-raystructureofoneofthenativeenzymesAdditionalmaterialonEisenberg’s3-DprofilemethodRef:Science(1991),253:164-170

Schematicdescriptionoftheconstructionofa3DstructureprofileSchematicdescriptionoftheconstructionofa3DstructureprofileSchematicdescriptionoftheconstruc