PREFOLDING AND PROTEIN FOLDING

预折叠和蛋白质折叠

• 批准号: 2824652
• 负责人: NICHOLAS COWAN
• 金额: $ 27.94万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2824652
• 关键词: Archaea; Escherichia coli; SDS polyacrylamide gel electrophoresis; biochemical evolution; cytoplasm; electron microscopy; enzyme activity; eukaryote; fungal proteins; molecular chaperones; myosins; polymerase chain reaction; polymerization; posttranslational modifications; protein binding; protein folding; protein metabolism; protein structure function; stress proteins; tissue /cell culture; yeasts

Chaperone proteins bind dynamically to non-native proteins and assist them during their folding, refolding, translocation and degradation. We have discovered a novel hexameric chaperone, prefoldin, which binds to non-native proteins and delivers them to chaperonin, to which it also binds. We propose to investigate the role of prefoldin in protein metabolism in the cytosol of eukaryotes. I. We will investigate a) the target range of prefoldin and b) the state of bound target protein. c) We will study the assembly of prefoldin subunits in vitro and during translation. d) We will determine whether prefoldin (and/or chaperonin) act on target proteins cotranslationally or posttranslationally or both, and where most folding of their major substrates (actin and tubulin) occurs. e) We will study the geometry of chaperonin binding and prefoldin/target protein binding by electron microscopy. f) We will examine the role of prefoldin in heatshock. g) We will test the hypothesis that prefoldin affect the partitioning of newly synthesized proteins among the chaperones present in the cell. This will be done by performing in vitro experiments with potentially competing chaperones including hsp70s, and altering the balance among these chaperones in vivo. h) We will examine the effect of prefoldin on the ATPase activity of chaperonin. II. We propose to study the role of prefoldin homologs from the archaea, to elucidate the structure, function and evolutionary history of this highly conserved chaperone. We will coexpress the two prefoldin subunit homologs from the archaeon Methanococcus jannaschii in E. coli a) for structural analysis and b) to explore their interaction with the corresponding chaperonin as well as their effect on the folding by and polymerization of the corresponding archaeal chaperonin. These studies are designed to throw light on the workings of prefoldin and the evolution of chaperone proteins.

伴侣蛋白动态地与非天然蛋白结合，并在其折叠、重折叠、移位和降解过程中协助它们。我们发现了一种新的六聚体伴侣蛋白，预折叠蛋白，它与非天然蛋白结合，并将它们输送到伴侣蛋白，它也与伴侣蛋白结合。我们建议研究前折叠蛋白在真核生物胞浆蛋白质代谢中的作用。我们将研究a)前折叠蛋白的靶范围和b)结合的靶蛋白的状态。C)我们将研究前折叠蛋白亚基在体外和翻译过程中的组装。D)我们将确定前折叠蛋白(和/或伴侣蛋白)是否以共翻译或翻译后或两者同时作用于靶蛋白，以及它们的主要底物(肌动蛋白和微管蛋白)的大部分折叠发生在哪里。E)我们将用电子显微镜研究伴侣蛋白结合和前折叠蛋白/靶蛋白结合的几何结构。F)我们将研究前折叠蛋白在热休克中的作用。G)我们将检验这一假设，即前折叠蛋白影响新合成的蛋白质在细胞中存在的伴侣之间的分配。这将通过对包括Hsp70在内的潜在竞争伴侣进行体外实验，并在体内改变这些伴侣之间的平衡来实现。H)我们将检测前折叠蛋白对伴侣蛋白ATPase活性的影响。II.我们建议研究古生菌中前折叠蛋白同源物的作用，以阐明这种高度保守的伴侣蛋白的结构、功能和进化史。我们将在大肠杆菌中共表达詹纳斯氏甲烷球菌的两个前折叠蛋白亚基同源物，用于结构分析和b)探索它们与相应的伴侣蛋白的相互作用以及它们对相应的古生菌伴侣蛋白的折叠和聚合的影响。这些研究旨在阐明前折叠蛋白的工作原理和伴侣蛋白的进化。