PREFOLDING AND PROTEIN FOLDING

预折叠和蛋白质折叠

• 批准号: 6181418
• 负责人: NICHOLAS COWAN
• 金额: $ 26.43万
• 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/6181418
• 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.

伴侣蛋白在折叠，重折叠，易位和降解过程中动态与非母蛋白的动态结合。 我们已经发现了一种新型的六聚体伴侣蛋白，该蛋白与非母蛋白结合并将其交付给伴侣蛋白，并与其结合。 我们建议研究蛋白质在蛋白质代谢中的作用在真核生物的胞质溶胶中。 I.我们将研究a）预卵素和b）结合靶蛋白的靶范围。 c）我们将在体外和翻译过程中研究前蛋白亚基的组装。 d）我们将确定前蛋白（和/或伴侣蛋白）是否在靶蛋白上作用于转倾上或翻译后或两者兼而有之，以及其主要底物（肌动蛋白和小管蛋白）的大多数折叠。 e）我们将研究通过电子显微镜结合伴侣蛋白结合和前蛋白/靶蛋白结合的几何形状。 f）我们将检查前蛋白在热休克中的作用。 g）我们将检验以下假设：预卵素会影响细胞中存在的伴侣蛋白之间新合成蛋白的分配。 这将通过对包括HSP70在内的潜在竞争伴侣进行体外实验，并改变体内这些伴侣之间的平衡来完成。 h）我们将检查前蛋白素对伴侣蛋白ATPase活性的影响。 ii。我们建议研究先天性同源物的作用，以阐明这种高度保守的伴侣的结构，功能和进化史。 我们将从大肠杆菌中的jannaschii中共表达两个前蛋白亚基同源物a）进行结构分析和b）探索它们与相应的chaperon伴侣蛋白的相互作用，以及它们对相应的Archaeal Chaperonin的折叠的影响。 这些研究旨在阐明预卵素的起作用和伴侣蛋白的进化。