MECHANISM OF ACTION OF THE HSP100 CHAPERONE CLPA

描述（申请人描述）：拟定研究的目的是 提供ATP依赖性解折叠的机制性理解， HSP 100分子伴侣环结构介导的蛋白水解 圆柱形蛋白酶，类似于19S“帽”组件的作用， 指导蛋白质被真核20S蛋白酶体圆柱体降解。 特别是，我们正在研究细菌HSP 100 分子伴侣，CIpA，一种六聚体环，在其每个环中具有两个ATP结合结构域， 84 kDa亚基，介导蛋白质底物的ATP依赖性解折叠， 使它们易位到同轴结合的 同源蛋白酶，ClpP，相同的23 kDa的堆叠双环十四聚体 丝氨酸蛋白酶亚基。几种模型基质的易位将是 研究使用荧光动力学测量，评估是否 未折叠的蛋白质被定向易位到ClpP中。的假设 蛋白水解直到底物移位完成才开始， 使用融合蛋白进行测试。ClpA上的底物结合位点将是 通过EM分析与ClpAP结合的金标记底物蛋白确定 配合物ClpA在定向解折叠中的ATP定向动态运动 和易位也将进行研究，使用快速冷冻和冷冻电镜 检查各种核苷酸状态的ClpAP复合物。结构功能 分析将进行，采用随机和定点突变的 ClpA，在体内和体外，研究功能，和晶体学 研究各种形式的ClpA以获得高分辨率结构 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.