Conformational cycles of molecular chaperones

描述(申请人提供)：HSP90是一种独特的伴侣蛋白，在真核生物中是必不可少的，它有助于产生和维持一组具有生物和医学意义的重要底物/客户的活性状态，包括许多信号转导蛋白。通过这些客户，Hsp90参与了衰老、信号转导和进化等生物过程。HSP90的功能需要ATP的水解以及客户和众多辅助伴侣的动态结合和释放。这种动态的大分子组装过程是许多关键生物学过程的基础，包括DNA复制和转录的启动。了解Hsp90的构象动力学将有助于深入了解其他动态的大分子复合体，并确定伴侣蛋白在信号转导中的作用。基于Hsp90的生化性质，Hsp90可能有许多不同的构象循环。我们正在阐明体内与生物相关的Hsp90构象。我们使用蛋白质工程策略来热力学稳定不同构象的Hsp90，以确定它们的生化性质和在体内的功能。这些实验的结果将描绘出激活体内客户的Hsp90构象，并确定生物学上相关的Hsp90伴侣周期。结合我们的体内研究，我们正在开发FRET实验，以监测客户成熟过程中Hsp90构象变化的动力学。HSP90是一种结构灵活的同源二聚体，它包含两个二聚化结构域：C结构域在生理浓度下主要是二聚体，而N结构域是ATP水解点并形成瞬时二聚体，这一应用有两个目的：(1)确定N-结构域结合在Hsp90伴侣循环和底物激活中的作用；(2)阐明Hsp90各亚基在底物激活过程中的功能。体内实验和蛋白质工程以及热力学和动力学分析的强大结合将为Hsp90的机制提供独特的见解。与公共健康相关：Hsp90蛋白是一种伴侣蛋白，帮助许多医学上重要的蛋白实现其最终的活性形状。这些医学上重要的蛋白质与衰老以及囊性纤维化和癌症等人类疾病有关。了解Hsp90的分子机制(这一应用的目标)，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.