STRUCTURE-FUNCTION STUDIES OF 70 KD HEAT SHOCK PROTEINS

70千道尔顿的热休克相关蛋白(HSP70相关)是广泛的- 传播开来，对细胞存活至关重要。他们看起来很亲密 涉及蛋白质的折叠、靶向和成熟等过程。 细菌和寄生虫HSP70相关蛋白具有高度的免疫原性；宿主 对这些高度保守的蛋白质的免疫反应可能会导致 自身免疫性综合症。 这项建议集中在一个问题上：什么是生化功能 热休克蛋白70相关蛋白在细胞中的分布？总体战略是结构化的-- 在分子水平上进行功能研究。具体目标是： 1.用X射线结晶学和原位杂交技术阐明了ATPase的作用机制。 特定的突变作用。 2.寻找证据支持或反对分子的主要构象变化 利用小角度溶液对蛋白质进行x射线散射。 3.划定底物识别内的底物结合功能 牛热休克同源蛋白(HSC70)的结构域，以网状蛋白为 底物，通过构建(I)C末端缺失突变体和(Ii) HSC70和另一种HSP70相关蛋白的嵌合体蛋白， 毕普。 4.探讨结构和顺序的功能意义 利用位点特异性技术比较ATPase片段和肌动蛋白的相似性 结合ATPase和Cathrin去包被活性的诱变 化验。 5.研究在HSC70 ATPase上使用劳厄衍射的可行性 片段来确定在此过程中进行的反应的结构 催化作用。 6.尝试结晶完整的HSP70相关蛋白和/或其 底物识别结构域。

The 70 kilodalton heat shock-related proteins (HSP70-related) are wide- spread and essential for cell viability. They appear to be intimately involved the processes of protein folding, targeting and maturation. Bacterial and parasite HSP70-related proteins are highly immunogenic; host immune responses to these highly conserved proteins may lead to autoimmunity syndromes. This proposal focuses on the question: What are the biochemical functions of the HSP70-related proteins in cells? The general strategy is structure- function studies at the molecular level. The specific aims are: 1.Elucidate the ATPase mechanism using x-ray crystallography and site- specific mutagenesis. 2.Search for evidence for or against a major conformational change in the proteins using small-angle solution x-ray scattering. 3.Delimit the substrate binding functions within the substrate recognition domain of the bovine heat shock cognate protein (HSC70), using clathrin as a substrate, by constructing (i) C-terminal deletion mutants and (ii) proteins which are chimeras of HSC70 and another HSP70 related protein, BiP. 4.Probe the functional significance of the structural and sequence similarities of the ATPase fragment and actin, using site-specific mutagenesis in conjunction with ATPase and clathrin uncoating activity assays. 5.Examine the feasibility of using Laue diffraction on the HSC70 ATPase fragment to determine the structures of the reaction intermediated during catalysis. 6.Attempt to crystallize intact HSP70-related proteins and/or their substrate recognition domains.