Functional Analysis of a Protein folding Catalyst

描述（由申请人提供）：二硫键的形成对于大多数分泌蛋白的正确折叠至关重要，包括许多具有药理意义的蛋白。近年来，对体内二硫氧化机理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。这为对二硫键形成过程进行广泛的生化和机理分析开辟了道路。DsbA是分泌蛋白的二硫化物的直接供体。然后它被DsbB再氧化。DsbB是已知的第一个利用醌的氧化能力产生二硫化物的酶；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了解二硫化物是如何产生的。DsbB结合并还原醌类；DsbB也与DsbA结合，但很可能通过硫醇二硫交换反应将其氧化。我建议研究DsbB如何与醌类和DsbA相互作用。在一种互补的方法中，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-醌识别。DsbA需要非常明确地识别DsbB，但它也需要能够识别相对广泛的不同折叠蛋白。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。我们还必须明确界定和区分DsbB中参与醌和DsbA相互作用的残基。为了实现这些目标，我们将利用强大的基因选择，体外进化，生化分析，结构研究和抑制性醌类似物的使用。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和醌相互反应的速率，来追踪从醌到DsbB再到DsbA的氧化还原当量的流动。我们的工作将有助于阐明二硫键形成的机制，这是蛋白质折叠的一个重要过程，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.