错误折叠与蛋白质构象病misfoldingandproteinconformational课件

ProteinfoldingProteinfolding1JamesDeweyWasonFrancisHarryComptonCrickJamesDeweyWason2DNARNAProein?DNARNAProein?3中国启动人类肝脏蛋白质组计划国际人类蛋白质组计划的２０％以上的任务。proteomics中国启动人类肝脏国际人类蛋白质组计划的２０％以上的任务。pr4为什么要开展

蛋白质折叠的研究?为什么要开展

蛋白质折叠的研究?5研究的源动力

disease免疫病毒研究的源动力disease免病毒6错误折叠与蛋白质构象病misfoldingandproteinconformational课件7Proteinfolding

&liveProteinfolding

&live8蛋白质研究先驱吴宪

1893,11,24---1959,8,8蛋白质研究先驱吴宪

1893,11,24---19599１９１２年到美国麻省理工学院。因愤于我国海军落后，初学造船工程。因受赫胥黎“生命的物理基础”一文的影响，２年后改习化学。１９１６年毕业，获理学学士学位，留校任化学系助教。１９１７年进哈佛大学医学院生物系，成为美国著名生物化学家Folin教授的研究生，进行血液化学研究。

1924年起用各种方法使蛋白质变性，

1931年得出如下理论：蛋白质的变性是由于蛋白质分子由折叠而变为舒展。１９１２年到美国麻省理工学院。因愤于我国海军落后，初学造船工10变性剂巯基乙醇复性ribonuclease

变性剂巯基复性ribonuclease11Somedenaturedproteins

canberenatured

denaturedmoleculeAnfinsen原理1961Somedenaturedproteins

can12Probabilitythatcorrectfoldingwouldoccurinribonuclease

giventhatthereare

8

cysteineresidues1/7x1/5x1/3x1=1/105expectedactivity

~1%observedactivitywas

100%Probabilitythatcorrectfoldi13ifoneconformationisexploredevery0.1psec,thentimetorefold(t)=1087sec2ntorsionanglescanhave32n~10n

possibleconformationsdirectedpathwaysoffoldingmustexistsifn=100,thennumberofconformations,10100

Mechanismstoexplainre-foldingFactorsdrivingproteinfoldingifoneconformationisexplore14UnfoldedstateFormationofelementsof2-stru.FrameworkmodelFoldedcon.Assemblyof2-stru.UnfoldedFormationofFramework15gabcdefgabcdefgabcdefgabcdefgabcdSTHMKQLEDKVEELLSKNYHLENEVARLKKLVGERGCN4leucinezippergabcdefgabcdefgabcdefga16错误折叠与蛋白质构象病misfoldingandproteinconformational课件17CDspectraofGCN4leucinezipperinthepresenceofdifferentconcentrationsofSDSSDS4MGuHClCDspectraofGCN4leucinezip18Changesofellipticityat222nminthepresenceofdifferentconcentrationsofSDSChangesofellipticityat22219NativegelelectrophoresisofleucinezippertreatedwithSDSofdifferentconcentrationsLane1wasthenativeleucinezipperpeptide(control);lanes2-6weresamplestreatedwith0.1,0.2,0.3,0.6,and1.0mMSDSNativegelelectrophoresisof20someleadstraightdownhillEnergysurfacestovisualizeproteinfoldingpathwaysAamorerealisticenergylandscapetheproteinisfunneledtowardsanativestatemanypathwaysarepossibleothersmayleadtoenergyminimathatdelayproperfoldingsomeleadstraightdownhillEne21ChangesoffluorescenceemissionspectraofTgdenaturedinvariousconcentrationsofGuHClThyroglobulinChangesoffluorescenceemissi22ANSbindingcharacteristicsofTginvariousGuHClconcentrationsANSbindingcharacteristicsof23蛋白质功能区蛋白质功能区24肌酸激酶活性部位荧光探针暴露的速度常数盐酸胍（M）荧光OPTA内源荧光失活k1k2k1k20.30.380.0490.010.00383.60.0031.02.90.044.3肌酸激酶活性部位荧光探针暴露的速度常数荧光OPTA失活k1k25酶活性部位

的柔性学说邹承鲁酶活性部位

的柔性学说邹承鲁26Protein–proteininterfacedesignProtein–protein27erythropoietinerythropoietin28错误折叠与蛋白质构象病misfoldingandproteinconformational课件29EPO-EPORERPH1-EPORLiuS,LiuSY,ZhuXL,LiangHH,CaoAN,ChangZJandLaiLH*.Nonnaturalprotein-proteininteraction-pairdesignbykeyresiduesgrafting.PNAS,2007,104,5330EPO-EPORERPH1-EPORLiuS,LiuSY,Z30药物研究药物研究31

抗氧化剂对A1-40结构的影响庾照学等,中国病理生理杂志,2000,16FT-IRspectraofA1-40inPBS(pH7.4)for30minFT-IRspectraofA1-40inPBS(pH7.4)for7days抗氧化剂对A1-40结构的影响FT-IRspect32FT-IRspectraofA1-40inPBS(pH7.4)for7daysFT-IRspectraofA1-40

inPBS(pH7.4)withTA9901for7days(percentratio:A1–40:TA9901=1∶1)FT-IRspectraofA1-40in33衰减全反射红外光谱研究人乳腺癌组织A1635/A1652A1625/A1652A1645/A1652A1662/A1652A1682/A1652benign0.980.650.640.490.17malignant0.430.230.560.370.09衰减全反射红外光谱A1635/A1652A134开阔思路开阔思路35记忆合金棒矫正脊柱侧凸卢世璧(院士)HSP肿瘤疫苗记忆合金棒矫正脊柱侧凸卢世璧HSP肿瘤疫苗36分子伴侣

molecularchaperones

新生肽链的折叠分子伴侣

molecularchaperones新生肽链37Misfolding&ProteinConformationalDisordersMisfolding&ProteinConformat38疯牛病带给生命科学界的思考MadcowTSE疯牛病带给生命科学界的思考MadcowTSE39Alzheimer’sD.AmyloidProtein&TauproteinFamilialvisceralAmyloidosis

Lysozyme….ParkinsonD.α-synuclein

HuntingtonD.Glutamine-repeatPrionD.

Prionprotein

Sicklecellanaemia

HaemoglobinConformationalBrainsDisordersProteinConforma-tionalDisorders(PCD)Alzheimer’sD.AmyloidP40HumanPrionDiseasesSporadicform Creutzfeldt-Jakobdisease(CJD)Familial(inherited)form FamilialCJD Fatalfamilialinsomnia Gerstmann-Straussler-ScheinkersyndromeAcquired(transmitted)form IatrogenicCJD Kuru

NewVariantCJD(relatedtoMadCowDisease)HumanPrionDiseasesSporadicf41AnimalPrionDiseasesScrapie

SheepandgoatBovinespongiformencephalopathy(MadCowDisease)

CattleFelinespongiformencephalopathy

Cat(domesticcats,cheetahs,pumas)Transmissibleminkencephalopathy

MinkChronicwastingdisease

Muledeer,elkAnimalPrionDiseasesScrapie42朊病毒(Prion)病的共同特征临床表现:痴呆、共剂失调、震颤等症状病理学上的特点:大脑皮层的神经原细胞退化、空泡变性、死亡、消失，星状胶质细胞增生，蛋白酶抗性的PrP积聚，有时产生淀粉样斑朊病毒(Prion)病的共同特征临床表现:病理学上的特点:43PrionProteinGene(PRNP)---Locatedonchromosome20inhumans,chromosome2inmouse---EncodesaglycoproteinwithtwositesforN-linkedoligosaccharitesandaC-terminalGPIanchor---Highexpressioninbrain.Lowerexpressioninperipheraltissues---~10-15%ofallcasesarefamilial.About20mutationsarelinkedtofamilialdisease.PrionProteinGene(PRNP)---Lo44TheNobelPrizeinPhysiology

orMedicine1976

fortheirdiscoveriesconcerningnewmechanismsforthe

originanddisseminationofinfectiousdiseases

BaruchS.BlumbergD.CarletonGajdusekTheNobelPrizeinPhysiology

45StanleyB.Prusiner

TheNobelPrizeinPhysiology

orMedicine1997

forhisdiscoveryofPrions-anewbiologicalprincipleofinfectionStanleyB.Prusiner

TheNobel46DNARNAProteintranscriptiontranslationSequencestructurefunctionfolding二个中心法则1.Genetics:2.Protein:中心法则?DNARNA47Conformationaltransition:

fromalpha-helixrichtobeta-sheetrichPrPc

PrPscConformationaltransition:PrP48PrPcPrPscPrP27-30PrPcPrPscPrP27-3049ReferencesRogerH.Pain，MechanismsofProteinFoldingBengtNölting，ProeinFoldingKinetics–BiophysicalMethodsLeninger,PrinciplesofBiochemistry,WorthPublishing,MathewsandVanHolde,Biochemistry,BenjaminCummingsReferencesRogerH.Pain，Mechan50思考问题：1.蛋白质折叠中的”中心法则”？2.联系生物物理技术部分所学内容，哪些技术可以用来进行蛋白质折叠研究，其根据是什么？思考问题：1.蛋白质折叠中的”中心法则”？51ProteinfoldingProteinfolding52JamesDeweyWasonFrancisHarryComptonCrickJamesDeweyWason53DNARNAProein?DNARNAProein?54中国启动人类肝脏蛋白质组计划国际人类蛋白质组计划的２０％以上的任务。proteomics中国启动人类肝脏国际人类蛋白质组计划的２０％以上的任务。pr55为什么要开展

蛋白质折叠的研究?为什么要开展

蛋白质折叠的研究?56研究的源动力

disease免疫病毒研究的源动力disease免病毒57错误折叠与蛋白质构象病misfoldingandproteinconformational课件58Proteinfolding

&liveProteinfolding

&live59蛋白质研究先驱吴宪

1893,11,24---1959,8,8蛋白质研究先驱吴宪

1893,11,24---195960１９１２年到美国麻省理工学院。因愤于我国海军落后，初学造船工程。因受赫胥黎“生命的物理基础”一文的影响，２年后改习化学。１９１６年毕业，获理学学士学位，留校任化学系助教。１９１７年进哈佛大学医学院生物系，成为美国著名生物化学家Folin教授的研究生，进行血液化学研究。

1924年起用各种方法使蛋白质变性，

1931年得出如下理论：蛋白质的变性是由于蛋白质分子由折叠而变为舒展。１９１２年到美国麻省理工学院。因愤于我国海军落后，初学造船工61变性剂巯基乙醇复性ribonuclease

变性剂巯基复性ribonuclease62Somedenaturedproteins

canberenatured

denaturedmoleculeAnfinsen原理1961Somedenaturedproteins

can63Probabilitythatcorrectfoldingwouldoccurinribonuclease

giventhatthereare

8

cysteineresidues1/7x1/5x1/3x1=1/105expectedactivity

~1%observedactivitywas

100%Probabilitythatcorrectfoldi64ifoneconformationisexploredevery0.1psec,thentimetorefold(t)=1087sec2ntorsionanglescanhave32n~10n

possibleconformationsdirectedpathwaysoffoldingmustexistsifn=100,thennumberofconformations,10100

Mechanismstoexplainre-foldingFactorsdrivingproteinfoldingifoneconformationisexplore65UnfoldedstateFormationofelementsof2-stru.FrameworkmodelFoldedcon.Assemblyof2-stru.UnfoldedFormationofFramework66gabcdefgabcdefgabcdefgabcdefgabcdSTHMKQLEDKVEELLSKNYHLENEVARLKKLVGERGCN4leucinezippergabcdefgabcdefgabcdefga67错误折叠与蛋白质构象病misfoldingandproteinconformational课件68CDspectraofGCN4leucinezipperinthepresenceofdifferentconcentrationsofSDSSDS4MGuHClCDspectraofGCN4leucinezip69Changesofellipticityat222nminthepresenceofdifferentconcentrationsofSDSChangesofellipticityat22270NativegelelectrophoresisofleucinezippertreatedwithSDSofdifferentconcentrationsLane1wasthenativeleucinezipperpeptide(control);lanes2-6weresamplestreatedwith0.1,0.2,0.3,0.6,and1.0mMSDSNativegelelectrophoresisof71someleadstraightdownhillEnergysurfacestovisualizeproteinfoldingpathwaysAamorerealisticenergylandscapetheproteinisfunneledtowardsanativestatemanypathwaysarepossibleothersmayleadtoenergyminimathatdelayproperfoldingsomeleadstraightdownhillEne72ChangesoffluorescenceemissionspectraofTgdenaturedinvariousconcentrationsofGuHClThyroglobulinChangesoffluorescenceemissi73ANSbindingcharacteristicsofTginvariousGuHClconcentrationsANSbindingcharacteristicsof74蛋白质功能区蛋白质功能区75肌酸激酶活性部位荧光探针暴露的速度常数盐酸胍（M）荧光OPTA内源荧光失活k1k2k1k20.30.380.0490.010.00383.60.0031.02.90.044.3肌酸激酶活性部位荧光探针暴露的速度常数荧光OPTA失活k1k76酶活性部位

的柔性学说邹承鲁酶活性部位

的柔性学说邹承鲁77Protein–proteininterfacedesignProtein–protein78erythropoietinerythropoietin79错误折叠与蛋白质构象病misfoldingandproteinconformational课件80EPO-EPORERPH1-EPORLiuS,LiuSY,ZhuXL,LiangHH,CaoAN,ChangZJandLaiLH*.Nonnaturalprotein-proteininteraction-pairdesignbykeyresiduesgrafting.PNAS,2007,104,5330EPO-EPORERPH1-EPORLiuS,LiuSY,Z81药物研究药物研究82

抗氧化剂对A1-40结构的影响庾照学等,中国病理生理杂志,2000,16FT-IRspectraofA1-40inPBS(pH7.4)for30minFT-IRspectraofA1-40inPBS(pH7.4)for7days抗氧化剂对A1-40结构的影响FT-IRspect83FT-IRspectraofA1-40inPBS(pH7.4)for7daysFT-IRspectraofA1-40

inPBS(pH7.4)withTA9901for7days(percentratio:A1–40:TA9901=1∶1)FT-IRspectraofA1-40in84衰减全反射红外光谱研究人乳腺癌组织A1635/A1652A1625/A1652A1645/A1652A1662/A1652A1682/A1652benign0.980.650.640.490.17malignant0.430.230.560.370.09衰减全反射红外光谱A1635/A1652A185开阔思路开阔思路86记忆合金棒矫正脊柱侧凸卢世璧(院士)HSP肿瘤疫苗记忆合金棒矫正脊柱侧凸卢世璧HSP肿瘤疫苗87分子伴侣

molecularchaperones

新生肽链的折叠分子伴侣

molecularchaperones新生肽链88Misfolding&ProteinConformationalDisordersMisfolding&ProteinConformat89疯牛病带给生命科学界的思考MadcowTSE疯牛病带给生命科学界的思考MadcowTSE90Alzheimer’sD.AmyloidProtein&TauproteinFamilialvisceralAmyloidosis

Lysozyme….ParkinsonD.α-synuclein

HuntingtonD.Glutamine-repeatPrionD.

Prionprotein

Sicklecellanaemia

HaemoglobinConformationalBrainsDisordersProteinConforma-tionalDisorders(PCD)Alzheimer’sD.AmyloidP91HumanPrionDiseasesSporadicform Creutzfeldt-Jakobdisease(CJD)Familial(inherited)form FamilialCJD Fatalfamilialinsomnia Gerstmann-Straussler-ScheinkersyndromeAcquired(transmitted)form IatrogenicCJD Kuru

NewVariantCJD(relatedtoMadCowDisease)HumanPrionDiseasesSporadicf92AnimalPrionDiseasesScrapie

SheepandgoatBovinespongiformencephalopathy(MadCowDisease)

CattleFelinespongiformencephalopathy

Cat(domesticcats,cheetahs,pumas)Transmissibleminkencephalopathy

MinkChronicwastingdisease

Muledeer,elkAnimalPrionDiseasesScrapie93朊病毒(Prion)病的共同特征临床表现:痴呆、共剂失调、震颤等症状病理学上的特点:大脑皮层的神经原细胞退化、空泡变性、死亡、消失，星状胶质细胞增生，蛋白酶抗性的PrP积聚，有时产生淀粉样斑朊病毒(Prion)病的共同特征临床表现:病理学上的特点:94PrionProteinGene(PRNP)---Locatedonchromosome20inhumans,chromosome2inmouse---EncodesaglycoproteinwithtwositesforN-linkedoligos