KINETICS AND MECHANISM OF THE HEAT SHOCK 70 PROTEIN DNAK

热休克 70 蛋白 DNAK 的动力学和机制

• 批准号: 2190104
• 负责人: Stephan N. Witt
• 金额: $ 9.61万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190104
• 关键词: Escherichia coli; adenosine triphosphate; bacterial proteins; chemical binding; chemical kinetics; circular dichroism; conformation; enzyme mechanism; enzyme substrate; fluorescence spectrometry; hydrolysis; intermolecular interaction; peptide structure; protein folding; proteolysis; stress proteins

Members of the 70-kD family of heat shock proteins (HSP70s) mediate protein-protein interactions, catalyzing protein folding, protein transport, the assembly of oligomeric proteins, and even the renaturation of heat-inactivated proteins-processes that are essential for life. Recent studies have revealed that HSP70s have a variety of important functions in the human immune system. Paradoxically, HSP70s are non- specific catalysts, that is, they appear to mediate protein-protein interactions by reacting in a relatively non-specific manner with unraveled segments of other proteins. The long-term objective of this research is to understand the molecular basis by which the heat shock-70 protein dnaK reacts non-specifically with other proteins, discriminating unfolded from folded proteins. Because the discrimination between folded and unfolded proteins is probably a kinetic effect, the specific aims to be pursued during this grant period involved elucidating the kinetics and mechanisms of three reactions-binding (P+dnaK-dnaK+P*), dissociation (dnaK-P*-dnaK+P) and ATP hydrolysis (nATP+dnaK+P*-nADP+dnaK+P), where P and P* represent non-fluorescent free labeled-peptide and fluorescent bound labeled-peptide (peptides are used to mimic the unraveled segments of other proteins). The first aim is to determine the rate-limiting step in the binding determine the mechanism of peptide dissociation from dnaK, to determine the effect of the peptide's structure on the rate of dissociation from dnaK, and to test the idea that there are multiple, overlapping interacting peptide binding site. The third aim is to understand how ATP-hydrolysis in the ATP-binding domain of dnaK is coupled to peptide dissociation from the peptide-binding domain. The fourth aim is to investigate the kinetics and mechanism of peptides binding to and dissociating from the peptide-binding domain fragment of dnaK, obtained by limited proteolysis of dnaK. The reactions between dnaK and fluorescently labeled-peptides will be followed using stopped- flow fluorescence spectrophotometry in conjunction with stopped-flow circular dichroism. Execution of these specific aims will provide quantitative information on the individual reaction steps involved in dnaK-catalyzed reactions.

70 kd热激蛋白（HSP70S）的成员介导 蛋白质蛋白质相互作用，催化蛋白质折叠，蛋白质 运输，寡聚蛋白的组装，甚至更新 对生命必不可少的热灭活的蛋白质处理。 最近的研究表明，HSP70具有多种重要 在人类免疫系统中的功能。 矛盾的是，HSP70是非 - 特定的催化剂，也就是说，它们似乎介导了蛋白质蛋白质 通过以相对非特异性的方式与 其他蛋白质的段。 这个长期目标 研究是了解热冲击70的分子基础 蛋白DNAK与其他蛋白质无反应，歧视 从折叠蛋白中展出。 因为折叠的歧视 而展开的蛋白质可能是一种动力学作用，具体的目的是 在此赠款期间被追捕涉及阐明动力学和 三种反应结合的机制（P+DNAK-DNAK+P*），解离 （DNAK-P*-DNAK+P）和ATP水解（NATP+DNAK+P*-NADP+DNAK+P），其中P P*代表非荧光的无标记肽和荧光 绑定的标记肽（肽用于模仿未分离的段 其他蛋白质）。 第一个目的是确定限速步骤 在结合中，确定肽与DNAK解离的机理， 确定肽结构对速率的影响 与DNAK解离，并测试有多个的想法， 重叠相互作用的肽结合位点。 第三个目标是 了解DNAK的ATP结合域中的ATP-Hydrolsys是如何 与肽结合结构域的肽分离结合。 这 第四目的是研究肽的动力学和机制 与肽结合结构域碎片结合并解离 DNAK，通过有限的DNAK蛋白水解获得。 之间的反应 使用停止的DNAK和荧光标记的肽 流量荧光分光光度法与停止流相结合 循环二科运动。 这些特定目标的执行将提供 有关涉及单个反应步骤的定量信息 DNAK催化反应。