Conformational cycles of molecular chaperones

HSP90是一种独特的伴侣蛋白，在真核生物中是必不可少的，有助于产生和维持活性的 一组精选的生物和医学上重要的底物/客户的状态，包括许多信号 转导蛋白。通过这些客户，Hsp90参与了生物过程，包括衰老、信号 转导和进化。HSP90的功能需要ATP的水解和HSP90的动态结合和释放 客户和众多的共同监护人。这种动态的大分子组装过程是许多 关键的生物学过程，包括DNA复制和转录的启动。了解 Hsp90的构象动力学将为研究其他动态的大分子络合物和 确定伴侣在信号转导中的作用。Hsp90的许多不同的构象周期是 根据Hsp90的生化特性，这是可能的。我们正在阐明与生物相关的Hsp90 活体内的构象。我们使用蛋白质工程策略来热力学稳定Hsp90在不同的 以确定它们的生化性质和在体内的功能。这些研究的结果 实验将描绘在体内激活客户的Hsp90构象，并从生物学上确定 相关的Hsp90分子伴侣周期。结合我们的活体研究，我们正在开发FRET实验 目的：监测客户成熟过程中热休克蛋白90构象的动态变化。HSP90在结构上是一种 含有两个二聚化结构域的灵活的同源二聚体：C-结构域主要是二聚体 生理浓度，而N-结构域是ATP水解的位置并形成瞬时二聚体， 这项提议有两个目的：(1)确定N-结构域关联在Hsp90伴侣环中的作用 和底物的激活，以及(2)阐明Hsp90各亚基在激活过程中的功能 底物。体内实验和蛋白质工程的强大结合 热力学和动力学分析将提供对Hsp90机制的独特见解。

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 proposal: (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 activating 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.