Protein folding

James Dewey Wason Francis Harry Compton Crick

DNA RNA Proein ?

proteomics 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。

为什么要开展 蛋白质折叠的研究?

disease 研究的源动力 病毒 免 疫

Protein folding & live

吴 宪 1893,11,24---1959,8,8 蛋白质研究先驱

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。

ribonuclease 变性剂 巯基 乙醇 复性

Some denatured proteins can be renatured Anfinsen原理 1961 Some denatured proteins can be renatured denatured molecule

Probability that correct folding would occur in ribonuclease given that there are 8 cysteine residues 1/7 x 1/5 x 1/3 x 1 = 1/105 expected activity ~ 1% observed activity was 100%

Mechanisms to explain re-folding Factors driving protein folding 2n torsion angles can have 32n ~ 10n possible conformations if n =100, then number of conformations, 10100 if one conformation is explored every 0.1 psec, then time to refold (t) = 1087 sec directed pathways of folding must exists

Framework model Formation of Assembly elements of 2-stru. of 2-stru. Folded con. Unfolded state

GCN4 leucine zipper gabcdefgabcdefgabcdefgabcdefgabcd STHMKQLEDKVEELLSKNYHLENEVARLKKLVGER

CD spectra of GCN4 leucine zipper in the presence of different concentrations of SDS 4 M GuHCl SDS

Changes of ellipticity at 222 nm in the presence of different concentrations of SDS

Native gel electrophoresis of leucine zipper treated with SDS of different concentrations Lane 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 SDS

Energy surfaces to visualize protein folding pathways a more realistic energy landscape A the protein is funneled towards a native state many pathways are possible some lead straight downhill others may lead to energy minima that delay proper folding

Changes of fluorescence emission spectra of Tg denatured in various concentrations of GuHCl Thyroglobulin

ANS binding characteristics of Tg in various GuHCl concentrations

蛋白质功能区

肌酸激酶活性部位荧光探针暴露的速度常数 盐酸胍 （M） OPTA 失活 k1 k2 0.3 0.38 0.049 0.015 0.5 内源荧光 失活 k1 k2 0.3 0.38 0.049 0.015 0.5 1.18 0.11 0.0038 3.6 0.003 1.0 2.9 0.04 4.3

酶活性部位 的柔性学说 邹承鲁

Protein–protein interface design

erythropoietin

EPO-EPOR ERPH1-EPOR LiuS,LiuSY,ZhuXL, LiangHH,CaoAN,ChangZJandLaiLH*. Nonnaturalprotein-proteininteraction-pairdesignbykeyresiduesgrafting. PNAS,2007,104,5330

药物研究

抗氧化剂对A1-40结构的影响 庾照学等,中国病理生理杂志,2000,16 FT - IR spectra of A1 - 40 in PBS(pH7. 4) for 30min FT - IR spectra of A1 - 40 in PBS(pH7. 4) for 7 days

FT- IR spectra of A1 - 40 in PBS(pH7 FT- IR spectra of A1 - 40 in PBS(pH7. 4) with TA9901 for 7 days (percent ratio : A1 – 40 :TA9901 = 1∶1) FT - IR spectra of A1 - 40 in PBS(pH7. 4) for 7 days

衰 减 全 反 射 红 外 光 谱 研究人乳腺癌组织 benign 0.98 0.65 0.64 0.49 0.17 malignant A1635/A1652 A1625/A1652 A1645/A1652 A1662/A1652 A1682/A1652 benign 0.98 0.65 0.64 0.49 0.17 malignant 0.43 0.23 0.56 0.37 0.09

开阔思路

记忆合金棒矫正脊柱侧凸 HSP 肿瘤疫苗 卢世璧 (院士)

分子伴侣 molecular chaperones 新生肽链的折叠 分子伴侣 molecular chaperones

Misfolding & Protein Conformational Disorders

疯牛病带给生命科学界的思考 Mad cow TSE

Alzheimer’s D. Amyloid  Protein & Tau protein Conformational Brains Disorders Parkinson D. α-synuclein Huntington D. Glutamine-repeat Protein Conforma- tional Disorders (PCD) Prion D. Prion protein Sickle cell anaemia Haemoglobin Familial visceral Amyloidosis Lysozyme ….

Human Prion Diseases Sporadic form Creutzfeldt-Jakob disease (CJD) Familial (inherited) form Familial CJD Fatal familial insomnia Gerstmann-Straussler-Scheinker syndrome Acquired (transmitted) form Iatrogenic CJD Kuru New Variant CJD (related to Mad Cow Disease)

Animal Prion Diseases Scrapie Sheep and goat Bovine spongiform encephalopathy (Mad Cow Disease) Cattle Feline spongiform encephalopathy Cat (domestic cats, cheetahs, pumas) Transmissible mink encephalopathy Mink Chronic wasting disease Mule deer, elk

朊病毒(Prion)病的共同特征 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 临床表现: 痴呆、共剂失调、震颤等症状

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.

Baruch S. Blumberg D. Carleton Gajdusek The Nobel Prize in Physiology or Medicine 1976 for their discoveries concerning new mechanisms for the origin and dissemination of infectious diseases Baruch S. Blumberg D. Carleton Gajdusek

Stanley B. Prusiner The Nobel Prize in Physiology or Medicine 1997 for his discovery of Prions - a new biological principle of infection

二个中心法则 DNA RNA Protein Sequence structure function 1. Genetics: 中心法则? transcription translation 中心法则? 2. Protein: Sequence structure function folding

Conformational transition: from alpha-helix rich to beta-sheet rich PrPc PrPsc

PrPsc PrP27-30 PrPc

References Roger H.Pain， Mechanisms of Protein Folding Bengt Nölting， Proein Folding Kinetics –Biophysical Methods Leninger, Principles of Biochemistry,Worth Publishing, Mathews and Van Holde, Biochemistry, Benjamin Cummings

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？