STRUCTURAL STUDIES OF AAA ATPASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白质必须正确折叠才能实现生物功能。与此同时，蛋白质聚集和错误折叠是许多毁灭性人类疾病的关键因素，例如阿尔茨海默病和普恩病毒介导的感染。与其他更传统的分子伴侣不同，酪蛋白水解酶B(ClpB)及其酵母同系物热休克蛋白104(Hsp104)具有从先前聚集的状态中拯救蛋白质的显著能力。ClpB/Hsp104家族的成员形成约600 kDa的六聚环结构，将ATP结合和水解产生的化学能转化为机械功。这项研究的目的是提供一个详细的机制了解ClpB和Hsp104如何促进先前聚集的蛋白质的解离。 项目1：ClpB六聚体的结构 ClpB是一种依赖于ATP的分子机器，它形成一个600 kDa的六聚体环结构，属于CLP/Hsp100家族的AAA+蛋白。然而，与其他分子伴侣不同，ClpB既不促进蛋白质的前向折叠，也不阻止蛋白质的聚集。相反，ClpB具有从聚集状态中拯救受压力破坏的蛋白质的非凡能力；这是诱导耐热性所必需的功能。 虽然结构-功能关系已经开始被理解，但目前还不清楚ClpB如何将来自ATP结合和水解的能量转化为机械功。 项目2：Hsp104分子伴侣的结构 Hsp104首先被发现是酵母应激反应中的重要成分，是一种依赖于ATP的蛋白质重塑因子，与ClpB一样，它可以从先前聚集的状态中拯救应激损伤的蛋白质。虽然细菌ClpB具有这样做的能力，但潜在的机制是不同的。例如，有人提出Hsp104的第二个AAA结构域对于寡聚是必不可少的，并且对于底物结合是必需的，而对于ClpB则相反。为了提供对Hsp104的详细的机制理解，我们正在使用CryoEM来确定Hsp104在核苷酸结合和游离状态下的结构

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteins must fold correctly in order to attain biological function. Concurrently, protein aggregation and misfolding are key contributors to many devastating human diseases such as Alzheimer's disease and prion-mediated infections. Unlike other more conventional molecular chaperones, the caseinolytic protease B (ClpB) and its yeast homolog heat-shock protein 104 (Hsp104) have the remarkable ability to rescue proteins from a previously aggregated state. Members of the ClpB/Hsp104 family form hexameric ring structures of ~600 kDa in molecular weight and convert chemical energy derived from ATP-binding and hydrolysis into mechanical work. The goal of this research is to provide a detailed mechanistic understanding how ClpB and Hsp104 facilitate the dissociation of previously aggregated proteins. Project 1: Structure of the ClpB hexamer ClpB is an ATP-dependent molecular machine, which forms a 600-kDa hexameric ring structure and belongs to the Clp/Hsp100 family of AAA+ proteins. Unlike other molecular chaperones, however, ClpB neither promotes the forward folding nor prevents the aggregation of proteins. Instead, ClpB has the remarkable ability to rescue stress-damaged proteins from an aggregated state; a function that is essential for induced thermotolerance. While the structure-function relationship is beginning to be understood, it remains unclear how ClpB converts the energy derived from ATP binding and hydrolysis into mechanical work. Project 2: Structure of the Hsp104 molecular chaperone First discovered as an essential component in the yeast stress response, Hsp104 is an ATP-dependent protein-remodeling factor, which, like ClpB, can rescue stress-damaged proteins from a previously aggregated state. While the ability to do so is shared with bacterial ClpB, the underlying mechanism is different. For instance, it has been proposed that the second AAA domain of Hsp104 is essential for oligomerization and is required for substrate binding while the reverse is true for ClpB. To provide a detailed mechanistic understanding of Hsp104, we are using cryoEM to determine the structure of Hsp104 both in the nucleotide-bound and -free states