MECHANISM OF ACTION OF THE HSP100 CHAPERONE CLPA

描述(申请人的描述)：建议研究的目标是 为了提供对依赖于ATP的展开和 HSP100分子伴侣环结构介导的蛋白水解酶 柱状蛋白，类似于19世纪90年代“帽子”组装的作用 真核生物的20S蛋白酶体圆柱体引导蛋白质降解。 特别是，我们正在研究细菌HSP100的机制 伴侣，CIPA，一个六聚体环，每个环上都有两个ATP结合域 84 kDa亚基，介导ATP依赖的蛋白质底物的去折叠和 使它们移位到同轴绑定中并由其降解 同源蛋白水解酶，CIPP，23 kDa的堆叠双环十四聚体 丝氨酸蛋白酶亚基。几种模型底物的移位将是 使用荧光动力学测量进行研究，评估是否 未折叠的蛋白质被定向转运到ClpP中。假设 在底物转运完成之前，蛋白质分解不会开始 使用融合蛋白进行检测。底物与ClpA结合的位置将是 结合CIpAP的金标记底物蛋白的EM分析 复合体。ClpA在定向展开过程中的动态三磷酸腺苷导向运动 还将使用快速冷冻和冷冻EM来研究移位 不同核苷酸状态下CIpAP复合体的检测。结构-功能 将进行分析，使用随机和定点突变的 CIPA，体内和体外，研究功能和结晶学 研究各种形式的CIPA以获得高分辨率的结构 信息。

DESCRIPTION (applicant's description): The objective of the proposed studies is to provide a mechanistic understanding of ATP-dependent unfolding and proteolysis mediated by HSP100 chaperone ring structures in association with cylindrical proteases, resembling the action of 19S "cap" assemblies in directing proteins for degradation by the eukaryotic 20S proteasome cylinder. In particular, we are studying the mechanism by which the bacterial HSP100 chaperone, CIpA, a hexameric ring with two ATP binding domains in each of its 84 kDa subunits, mediates ATP dependent unfolding of protein substrates and commits them to translocation into and degradation by the coaxially bound cognate protease, CIpP, a stacked double ring tetradecamer of identical 23 kDa serine protease subunits. Translocation of several model substrates will be studied using fluorescence dynamics measurements, assessing whether the unfolded proteins are directionally translocated into ClpP. The hypothesis that proteolysis does not commence until substrate translocation is completed will be tested using fusion proteins. The site of substrate binding on ClpA will be determined by EM analysis of gold-tagged substrate proteins bound to CIpAP complexes. The dynamic ATP-directed movements of ClpA in directing unfolding and translocation will also be studied, using rapid-freezing and cryoEM examination of CIpAP complexes in various nucleotide states. Structure-function analyses will be carried out, employing random and site-directed mutants of CIpA, both in vivo and in vitro, to study function, and crystallographic studies of various forms of CIpA to obtain high-resolution structural information.