KINETICS AND MECHANISM OF THE HEAT SHOCK 70 PROTEIN DNAK

70-kD热休克蛋白家族成员(Hsp70)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，甚至复性 热灭活蛋白质--生命所必需的过程。 最近的研究表明，Hsp70具有多种重要的 在人体免疫系统中发挥作用。矛盾的是，Hsp70不是 特定的催化剂，也就是说，它们似乎是调节蛋白质-蛋白质 通过以相对非特定的方式与 解开了其他蛋白质的片段。这样做的长期目标是 研究是为了了解热休克-70的分子基础 蛋白质dnaK与其他蛋白质发生非特异性反应，区分 从折叠的蛋白质中解开。因为折叠和折叠之间的歧视 而未折叠的蛋白质可能是一种动力学效应，其具体目的是 在这一授权期内进行的研究包括阐明动力学和 三个反应的机理--结合(P+dNAK-dNAK+P*)、解离 (dNAK-P*-dNAK+P)和ATP水解(nATP+dNAK+P*-NADP+dNAK+P)，其中P 和P*代表非荧光自由标记肽和荧光 结合标记的多肽(多肽用于模拟未解开的片段 其他蛋白质)。第一个目标是确定速率限制步骤 在结合中确定了多肽从dNAK解离的机制， 为了确定多肽的结构对转移率的影响 从dNAK解离，并测试有多个， 重叠的相互作用肽结合部位。第三个目标是 了解dNAK的ATP结合域中的ATP水解酶是如何 偶联到多肽从多肽结合域解离。这个 第四个目的是研究多肽的动力学和机理。 与多肽结合结构域片段的结合和解离 DNAK，通过对dNAK进行有限的蛋白分解而获得。两国之间的反应 DNAK和荧光标记的多肽将使用停止- 流动荧光分光光度与停流联用 圆二向色性。这些具体目标的实现将提供 有关所涉及的各个反应步骤的定量信息 DNAK催化的反应。

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.