PROTEIN FOLDING IN THE EUKARYOTIC CYTOSOL

描述(改编自申请者的摘要)：蛋白质的方式 获得了迄今为止被认为是正确的三维结构 一个自发的过程。在许多情况下，蛋白质折叠需要 一种或多种其他蛋白质的作用，称为分子伴侣。这 该建议涉及促进蛋白质折叠的过程。 真核细胞胞浆。研究人员发现了一种细胞质伴侣蛋白 (类似于在原核生物和细胞器中发现的那些)凭借其 折叠变性肌动蛋白的能力；他们发现同样的伴侣蛋白， 与两个蛋白质辅助因子一起，也促进了α- 和β-微管蛋白。胞质促进折叠的机制 伴侣蛋白和附加辅助因子的功能作用是完全 未知。1)他将提纯释放所需的辅因子 天然的α-和β-微管蛋白。提纯的材料将用于 通过生化分析和测序确定其功能的实验 分析。2)出乎意料的是，调查人员的数据指向了一个选择性的 胞质伴侣蛋白与其有限亚群的相互作用 细胞质蛋白。他将尝试识别蛋白质(除了 肌动蛋白和微管蛋白)，由伴侣识别。3)他会的 进行实验以确定肌动蛋白和微管蛋白中的那些基序 负责由伴侣识别的多肽，a)通过 定义与之形成(或不形成)二元络合物的亚片段 伴侣，b)通过竞争实验来定义性质和 相互作用所需地点的特殊性，以及c)通过识别 被保护不被蛋白质降解的靶蛋白片段 它们与伴侣的相互作用。4)考恩博士将调查 ATP水解在胞质伴侣蛋白介导的折叠中的作用 反应。5)他将确定底物结合到 细胞质伴侣蛋白。6)他将提纯和鉴定细胞质 与新翻译形成复合体的因子(伴侣蛋白除外) 肌动蛋白和微管蛋白，并进行实验以确定这些蛋白的作用 在整个折叠途径中的因素。

DESCRIPTION (Adapted from the applicant's abstract): The way a protein acquires its correct three-dimensional structure was hitherto thought to be a spontaneous process. The protein folding in many cases requires the action of one or more other proteins termed molecular chaperones. This proposal concerns the process of facilitated protein folding in the eukaryotic cytosol. The investigator discovered a cytoplasmic chaperonin (analogous to those found in prokaryotes and organelles) by virtue of its ability to fold denatured actin; they find that the same chaperonin, together with two protein cofactors, also facilitates the folding of alpha- and beta-tubulin. The mechanism of facilitated folding by the cytoplasmic chaperonin and the functional role of additional cofactors are completely unknown. 1) He will purify the cofactors that are required for the release of native alpha- and beta-tubulin. Purified material will be used in experiments to determine their function via biochemical assays and sequence analysis. 2) Unexpectedly, the investigators data points to a selective interaction of the cytoplasmic chaperonin with a limited subset of cytoplasmic proteins. He will attempt to identify proteins (other than actin and tubulin) that are recognized by the chaperonin. 3) He will perform experiments to define those motifs within the actin and tubulin polypeptides that are responsible for recognition by the chaperonin, a) by defining subfragments that form (or fail to form) binary complexes with chaperonin, b) via competition experiments to define the nature and specificity of the sites required for interaction, and c) by identifying fragments of target protein that are protected from proteolysis by virtue of their interaction with chaperonin. 4) Dr. Cowan will investigate the role of ATP hydrolysis in the cytosolic chaperonin-mediated folding reaction. 5) He will determine the stoichiometry of substrate binding to the cytoplasmic chaperonin. 6) He will purify and characterize cytoplasmic factors (other than chaperonin) that form complexes with newly translated actin and tubulin, and perform experiments to define the role of these factors in the overall folding pathway.