STRUCTURE-FUNCTION STUDIES OF 70 KD HEAT SHOCK PROTEINS

70 KD 热休克蛋白的结构功能研究

• 批准号: 3297215
• 负责人: David B McKay
• 金额: $ 20.27万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-10-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297215
• 关键词: X ray crystallography; actins; active sites; adenosinetriphosphatase; chemical kinetics; chimeric proteins; clathrin; conformation; crystallization; enzyme mechanism; enzyme substrate; enzyme substrate complex; low angle X ray diffraction analysis; mutant; polymerase chain reaction; protein engineering; protein structure function; site directed mutagenesis; stress proteins

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.

70 千道尔顿热休克相关蛋白（HSP70 相关）广泛- 传播并对细胞活力至关重要。 他们看似亲密 涉及蛋白质折叠、靶向和成熟的过程。 细菌和寄生虫HSP70相关蛋白具有高度免疫原性；主持人 对这些高度保守的蛋白质的免疫反应可能会导致 自身免疫综合征。 该提案关注的问题是：什么是生化功能？ 细胞中的HSP70相关蛋白？ 总体策略是结构- 分子水平的功能研究。 具体目标是： 1.利用X射线晶体学和位点阐明ATPase机制 特异性诱变。 2.寻找支持或反对重大构象变化的证据 使用小角度溶液 X 射线散射的蛋白质。 3.界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，使用网格蛋白作为 底物，通过构建（i）C端缺失突变体和（ii） 蛋白质是HSC70和另一种HSP70相关蛋白质的嵌合体， 毕普。 4.探究结构和序列的功能意义 ATP酶片段和肌动蛋白的相似性，使用位点特异性 与 ATP 酶和网格蛋白脱壳活性结合的诱变 化验。 5.检查在HSC70 ATPase上使用Laue衍射的可行性 片段以确定中间反应的结构 催化。 6.尝试结晶完整的HSP70相关蛋白和/或其 底物识别域。