错误折叠与蛋白质构象病misfoldingandproteinconational课件.ppt

Protein foldingJames Dewey WasonFrancis Harry Compton Crick DNA RNA Proein?中国启动人类肝脏中国启动人类肝脏蛋白质组计划蛋白质组计划国际人类蛋白质组计划国际人类蛋白质组计划的以上的任务的以上的任务proteomics为什么要开展为什么要开展蛋白质蛋白质折叠折叠的研究的研究?研研究究的的源源动动力力 disease 免疫病毒Protein folding&live蛋白质研究先驱蛋白质研究先驱吴吴 宪宪 1893,11,24-1959,8,8年到美国麻省理工学院因愤于我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国海军落后，初学造船工程因受赫胥黎“生命的物理基础生命的物理基础”一文的影响，年后改习一文的影响，年后改习化学年毕业，获理学学士学位，化学年毕业，获理学学士学位，留校任化学系助教年进哈佛大学留校任化学系助教年进哈佛大学医学院生物系，成为美国著名生物化学家医学院生物系，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，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。

蛋白质分子由折叠而变为舒展变性剂变性剂巯基巯基乙醇乙醇复性复性ribonuclease Some denatured proteins can be renatured denatured moleculeAnfinsen原理原理 1961Probability that correct folding would occur in ribonuclease given that there are 8 cysteine residues 1/7 x 1/5 x 1/3 x 1=1/105expected activity 1%observed activity was 100%if one conformation is explored every 0.1 psec,then time to refold(t)=1087 sec2n torsion angles can have 32n 10n possible conformationsdirected pathways of folding must existsif n=100,then number of conformations,10100 Mechanisms to explain re-folding Factors driving protein foldingUnfolded stateFormation of elements of 2-stru.Framework modelFolded con.Assembly of 2-stru.g a b c d e f g a b c d e f g a b c d e f g a b c d e f g a b c dSTHMKQLEDKVEELLSKNYHLENEVARLKKLVGERGCN4 leucine zipper CD spectra of GCN4 leucine zipper in the presence of different concentrations of SDSSDS4 M GuHClChanges of ellipticity at 222 nm in the presence of different concentrations of SDSNative gel electrophoresis of leucine zipper treated with SDS of different concentrationsLane 1 was the native leucine zipper peptide(control);lanes 2-6 were samples treated with 0.1,0.2,0.3,0.6,and 1.0 mM SDSsome lead straight downhillEnergy surfaces to visualize protein folding pathwaysAa more realistic energy landscapethe protein is funneled towards a native statemany pathways are possibleothers may lead to energyminima that delay proper foldingChanges of fluorescence emission spectra of Tg denatured in various concentrations of GuHClThyroglobulinThyroglobulinANS binding characteristics of Tg in various GuHCl concentrations蛋白质蛋白质功能区功能区肌酸激酶活性部位荧光探针肌酸激酶活性部位荧光探针暴露的速度常数暴露的速度常数盐酸胍盐酸胍（M）荧光荧光OPTA内源荧内源荧光光失活失活k1k2k1k20.30.38 0.0490.0150.51.180.110.00383.60.0031.02.90.044.3酶活性部位酶活性部位的柔性学说的柔性学说邹承鲁邹承鲁 Proteinprotein interface design erythropoietin EPO-EPORERPH1-EPORLiuS,LiuSY,ZhuXL,LiangHH,CaoAN,ChangZJandLaiLH*.Nonnaturalprotein-proteininteraction-pairdesignbykeyresiduesgrafting.PNAS,2007,104,5330 药物研究药物研究 抗氧化抗氧化剂对A 1-40结构的影响构的影响庾照学等庾照学等,中国病理生理杂志中国病理生理杂志,2000,16FT-IR spectra of A 1-40 in PBS(pH7.4)for 30minFT-IR spectra of A 1-40 in PBS(pH7.4)for 7 daysFT-IR spectra of A 1-40 in PBS(pH7.4)for 7 daysFT-IR spectra of A 1-40 in PBS(pH7.4)with TA9901 for 7 days(percent ratio:A 1 40:TA9901=1 1)衰衰 减减 全全 反反 射射 红红 外外 光光 谱谱研究研究人乳腺癌组织人乳腺癌组织A1635/A1652A1625/A1652A1645/A1652A1662/A1652A1682/A1652benign0.980.650.640.490.17malignant0.430.230.560.370.09开阔思路开阔思路记忆合金棒矫正脊柱侧凸 卢世璧卢世璧(院士院士)HSP 肿瘤疫苗分子伴侣分子伴侣molecular chaperones 新生肽链的折叠新生肽链的折叠Misfolding&Protein Conformational Disorders疯牛病带给生命科学界的思考疯牛病带给生命科学界的思考Mad cowMad cowTSETSETSETSEAlzheimers D.Amyloid Protein&Tau proteinFamilial visceral Amyloidosis Lysozyme.Parkinson D.-synuclein Huntington D.Glutamine-repeatPrion D.Prion protein Sickle cell anaemia HaemoglobinConformational BrainsDisordersProteinConforma-tionalDisorders(PCD)Human Prion DiseasesSporadic formCreutzfeldt-Jakob disease(CJD)Familial(inherited)formFamilial CJDFatal familial insomniaGerstmann-Straussler-Scheinker syndromeAcquired(transmitted)formIatrogenic CJDKuruNew Variant CJD(related to Mad Cow Disease)Animal Prion DiseasesScrapieSheep and goatBovine spongiform encephalopathy(Mad Cow Disease)CattleFeline spongiform encephalopathyCat(domestic cats,cheetahs,pumas)Transmissible mink encephalopathy MinkChronic wasting diseaseMule deer,elk朊病毒(Prion)病的共同特征 临床表现临床表现:痴呆、共剂失调、震颤等症状痴呆、共剂失调、震颤等症状 病理学上的特点病理学上的特点:大脑皮层的神经原细胞退化、空泡变性、死亡、消失，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，蛋白酶抗性的星状胶质细胞增生，蛋白酶抗性的PrP积聚，有时产生淀积聚，有时产生淀粉样斑粉样斑 Prion Protein Gene(PRNP)-Located on chromosome 20 in humans,chromosome 2 in mouse-Encodes a glycoprotein with two sites for N-linked oligosaccharites and a C-terminal GPI anchor-High expression in brain.Lower expression in peripheral tissues-10-15%of all cases are familial.About 20 mutations are linked to familial disease.The Nobel Prize in Physiology or Medicine 1976for their discoveries concerning new mechanisms for the origin and dissemination of infectious diseasesBaruch S.BlumbergD.Carleton GajdusekStanley B.PrusinerThe Nobel Prize in Physiology or Medicine 1997for his discovery of Prions-a new biological principle of infectionDNA RNA ProteinDNA RNA ProteintranscriptiontranscriptiontranslationtranslationSequence structure functionSequence structure functionfoldingfolding二个中心法则二个中心法则1.Genetics:2.Protein:中心法则中心法则?Conformational transition:from alpha-helix rich to beta-sheet richPrPc PrPscPrPcPrPscPrP2。