Conformational cycles of molecular chaperones

描述（由申请人提供）：Hsp 90是一种独特的伴侣蛋白，在真核生物中是必需的，有助于产生和维持一组选定的生物学和医学上重要的底物/客户（包括许多信号转导蛋白）的活性状态。通过这些客户，Hsp 90参与了衰老、信号转导和进化等生物过程。热休克蛋白90的功能需要ATP水解和客户端和许多辅助分子伴侣的动态结合和释放。这种类型的动态大分子组装过程是许多关键生物过程的基础，包括DNA复制和转录的起始。了解热休克蛋白90的构象动力学将提供洞察到其他动态大分子复合物，并确定分子伴侣在信号转导中的作用。基于Hsp 90的生化性质，Hsp 90的许多不同构象循环是可能的。我们正在阐明生物学相关的热休克蛋白90在体内的构象。我们使用蛋白质工程策略，以不同的构象，以确定其生化特性和它们的功能在体内热休克蛋白90的免疫稳定。这些实验的结果将描绘热休克蛋白90的构象，激活客户在体内，并确定生物学相关的热休克蛋白90分子伴侣循环。结合我们的体内研究，我们正在开发FRET实验，以监测客户端成熟过程中的热休克蛋白90构象变化的动力学。Hsp 90是一种结构灵活的同源二聚体，含有两个二聚化结构域：C结构域在生理浓度下主要是二聚体，而N结构域是ATP水解的位点并形成瞬时二聚体。本申请有两个目的：（1）确定N-结构域缔合在Hsp 90分子伴侣循环和底物活化中的作用，（2）阐明各Hsp 90亚基在底物活化过程中的功能。体内实验和蛋白质工程与热力学和动力学分析的有力结合将为Hsp 90的机制提供独特的见解。Hsp 90蛋白是一种伴侣蛋白，可以帮助许多医学上重要的蛋白质达到其最终的活性形状。这些医学上重要的蛋白质与衰老以及包括囊性纤维化和癌症在内的人类疾病有关。了解Hsp 90的分子机制（本申请的目标）将为合理设计治疗这些人类疾病的药物提供生化蓝图。

DESCRIPTION (provided by applicant): Hsp90 is a unique chaperone that is essential in eukaryotes and that helps to produce and maintain the active state of a select set of biologically and medically important substrates/clients including many signal transduction proteins. Through these clients, Hsp90 is involved in biological processes including aging, signal transduction and evolution. Hsp90 function requires ATP hydrolysis and the dynamic binding and release of clients and numerous co-chaperones. This type of dynamic macromolecular assembly process underlies many critical biological processes including DNA replication and the initiation of transcription. Understanding the conformational dynamics of Hsp90 will provide insights into other dynamic macromolecular complexes and determine the role of chaperones in signal transduction. Many different conformational cycles of Hsp90 are possible based on the biochemical properties of Hsp90. We are elucidating the biologically relevant Hsp90 conformations in vivo. We use protein engineering strategies to thermodynamically stabilize Hsp90 in distinct conformations in order to determine their biochemical properties and their function in vivo. The results of these experiments will delineate the Hsp90 conformations that activate clients in vivo and determine the biologically relevant Hsp90 chaperone cycle. In conjunction with our in vivo studies, we are developing FRET experiments to monitor the kinetics of Hsp90 conformational changes during client maturation. Hsp90 is a structurally flexible homodimer that contains two dimerization domains: the C-domain is predominantly dimeric at physiologic concentration, while the N-domain is the site of ATP hydrolysis and forms transient dimers, There are two aims to this application: (1) to determine the role of N-domain association in the Hsp90 chaperone cycle and the activation of substrates, and (2) to elucidate the function of each Hsp90 subunit during the activation of substrates. The powerful combination of in vivo experiments and protein engineering together with thermodynamic and kinetic analyses will provide unique insight into the mechanism of Hsp90. PUBLIC HEALTH RELEVANCE: The Hsp90 protein is a chaperone that helps many medically important proteins to achieve their final active shape. These medically important proteins are involved in aging as well as human diseases including cystic fibrosis and cancer. Understanding the molecular mechanism of Hsp90 (the goal of this application), will provide a biochemical blueprint for the rational design of drugs to treat these human diseases.