Conformational cycles of molecular chaperones

热休克蛋白90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 选择一组生物学和医学上重要底物/客户的状态 转导蛋白通过这些客户端，Hsp 90参与生物过程，包括衰老，信号转导， transduction转导and evolution进化.热休克蛋白90的功能需要ATP水解和热休克蛋白90的动态结合和释放。 客户和众多的监护人这种类型的动态大分子组装过程构成了许多 关键的生物过程，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其他动态大分子复合物， 确定分子伴侣在信号转导中的作用。热休克蛋白90有许多不同的构象循环， 根据Hsp 90的生物化学特性，这是可能的。我们正在阐明生物学相关的Hsp 90 体内构象。我们使用蛋白质工程策略，在不同的细胞中稳定Hsp 90， 构象，以确定它们的生物化学性质和它们在体内的功能。的结果予以 实验将描绘Hsp 90构象，激活客户在体内，并确定生物学 相关的Hsp 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 监测客户成熟过程中Hsp 90构象变化的动力学。HSP 90是一种结构上 含有两个二聚化结构域的柔性同源二聚体：C结构域主要是二聚体， 生理浓度，而N-结构域是ATP水解的位点并形成瞬时二聚体， 本研究的目的有两个：（1）确定N-结构域结合在Hsp 90分子伴侣循环中的作用 和底物的活化，以及（2）阐明每个Hsp 90亚基在活化过程中的功能 印刷受体.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独特的洞察热休克蛋白90的机制。

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.