PREFOLDING AND PROTEIN FOLDING

伴侣蛋白与非天然蛋白动态结合，并在它们的折叠、再折叠、易位和降解过程中提供帮助。我们发现了一种新型的六聚体伴侣蛋白，预折叠蛋白，它与非天然蛋白质结合，并将它们传递给伴侣蛋白，伴侣蛋白也与之结合。我们拟探讨前折叠蛋白在真核生物胞浆中蛋白质代谢中的作用。1 .我们将研究a) prefoldin的靶点范围和b)结合靶蛋白的状态。c)我们将在体外和翻译过程中研究折叠蛋白前亚基的组装。d)我们将确定折叠前蛋白（和/或伴侣蛋白）是共翻译还是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）的大多数折叠发生在哪里。e)我们将通过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)我们将研究预折叠蛋白在热休克中的作用。g)我们将检验预折叠蛋白影响细胞中存在的伴侣蛋白之间新合成蛋白质的分配的假设。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)我们将检验前折叠蛋白对伴侣蛋白atp酶活性的影响。2。我们建议研究来自古细菌的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来自jannaschii甲烷球菌的两个前折叠蛋白亚基同源物a)进行结构分析，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细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。

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.