Functional Analysis of a Protein folding Catalyst

描述(由申请人提供)：二硫键的形成对大多数分泌蛋白质的正确折叠至关重要，包括许多药理上的重要作用。在过去的几年里，对体内二硫化物氧化机制的研究取得了很大进展。我们最近成功地建立了二硫键形成的途径。这为二硫键形成过程的广泛生化和机理分析开辟了道路。DsbA是分泌蛋白质的二硫化物的直接供体。然后它被DsbB重新氧化。DsbB是已知的第一种利用苯醌的氧化力从头产生二硫化物的酶；它的催化活性是细胞中二硫化物的主要来源。对DsbB机理的分析将使我们深入了解二硫化物是如何产生的。DsbB结合并还原苯二酚；DsbB也与DsbA结合，但它氧化DsbA，很可能是通过硫醇二硫键交换反应。我建议研究DsbB如何与苯二酚和DsbA相互作用。作为补充，我将研究DsbA如何识别它的两种底物：折叠蛋白和DsbB。这些问题的答案将使我们深入了解DsbA的伴侣作用、蛋白质-蛋白质相互作用以及蛋白质-苯醌识别。DsbA需要非常特别地识别DsbB，但它也需要能够识别相对较广的不同折叠蛋白的光谱。我们必须清楚地定义和区分DsbA中参与未折叠蛋白和DsbB相互作用的残基。我们还必须清楚地定义和区分DsbB中参与苯二酚和DsbA相互作用的残基。为了实现这些目标，我们将利用强大的遗传选择、体外进化、生化分析、结构研究和抑制性苯醌类似物的使用。为了研究DsbB的作用机制，我们将通过测量DsbB的不同结构域与DsbA和苯二酚相互反应的速率来追踪氧化还原当量从苯二酚通过DsbB再到DsbA的流动。我们的工作将有助于阐明二硫键的形成机制，这是蛋白质折叠的关键过程，并揭示蛋白质折叠因子如何能够识别相对广泛的部分折叠蛋白质。

DESCRIPTION (provided by applicant): The formation of DISULFIDE bonds is vital for the proper folding of most secreted proteins, including many of pharmacological importance. Great progress has been made in the last few years in understanding the mechanism of disulfide oxidation in vivo. We have recently succeeded in establishing the pathway of disulfide bond formation. This opens up the way for extensive biochemical and mechanistic analysis of the process of disulfide bond formation. DsbA acts as a direct donor of disulfides to secreted proteins. It then is reoxidized by DsbB. DsbB is the first enzyme known to use the oxidizing power of quinones to generate disulfides de novo; its catalytic activity is the primary source of disulfides in the cell. Analysis of DsbB's mechanism will give us insights as to how disulfides are created. DsbB binds and reduces quinones; DsbB also binds to DsbA but it oxidizes it, most likely via a thiol disulfide exchange reaction. I propose to study how DsbB interacts with quinones and DsbA. In a complementary approach, I will study how DsbA recognizes its two substrates: folding proteins and DsbB. The answer to these questions will give us insights into DsbA's chaperone action, protein-protein interactions as well as protein-quinone recognition. DsbA needs to very specifically recognize DsbB, but it also needs to be able to recognize a relatively wide spectrum of different folding proteins. We must clearly define and distinguish the residues in DsbA that are involved in unfolded protein and DsbB interaction. We must also clearly define and distinguish the residues in DsbB that are involved in quinone and DsbA interaction. To accomplish these goals, we will exploit powerful genetic selections, in vitro evolution, biochemical assays, structural studies and the use of inhibitory quinone analogues. To investigate the mechanism of DsbB action, we will trace the flow of redox equivalents from quinones through DsbB and then on to DsbA by measuring the rates at which various domains of DsbB react with each other, with DsbA and with quinones. Our work will help illuminate the mechanism of disulfide bond formation, a process vital for protein folding and reveal how protein folding factors are able to recognizes a relatively wide variety of partially folded proteins.