Genetics of Bacterial Thiol Redox Proteins

描述(由申请人提供)：本项目的目标是了解具有二硫键氧化还原酶活性的蛋白质的各种重要细胞活性的生理和分子基础。我们专注于细菌大肠杆菌中这一蛋白质家族的成员，包括硫氧还蛋白超家族。我们将通过以下方式探索这些蛋白质的不同作用：1)研究它们合成的调节，以确定它们参与的重要细胞反应；2)通过捕获这些蛋白质与其底物的混合二硫键复合体(这将是为了定义这些蛋白质的迄今未被检测到的底物)，以及3)分离缺失许多这些成分的菌株中的抑制子突变。我们预计，这些抑制基因突变的特征将揭示蛋白质家族的更多成员，并提供有关已知成员功能的新信息。我们还将确定硫氧还蛋白家族，其成员具有高度保守的三维结构，如何经常表现出如此不同的底物特异性。改变硫氧还蛋白1和硫氧还蛋白2之间的特异性差异的交换结构和突变将阐明这个问题。特异性突变突变的选择是基于硫氧还蛋白2不能还原蛋氨酸亚砜还原酶。遗传学研究将与通过与X射线结晶学家合作获得的结构信息相结合。我们将深入表征蛋白质DsbB的作用机制。DsbB是一种膜蛋白，它是硫醇氧化酶DsbA重新氧化所必需的，将其电子传递给奎宁。我们已经将这个过程分解为几个步骤。已经分离的DsbB突变体的集合将被用来确定蛋白质的不同结构域在这一复杂的一系列步骤中的作用。从合作的核磁共振工作和遗传研究中获得的蛋白质结构信息将与这种突变分析结合起来，以了解这种蛋白质的功能。对二硫键形成和还原途径的研究已经为增加抗体和组织纤溶酶原激活剂等医学上重要蛋白质的产生提供了好处，这项研究应该为这些努力提供更多的信息。此外，这些蛋白质在许多细胞过程中扮演着重要的角色，无论是正常的还是病理的。

DESCRIPTION (provided by applicant): The goal of this project is to understand the physiological and molecular basis for the wide variety of important cellular activities of proteins with disulfide bond oxidoreductase activity. We focus on the members of this family of proteins in the bacteria E. coli, including the thioredoxin superfamily. We will explore the differing roles of these proteins by 1) studying the regulation of their synthesis in order to define important cellular responses they are involved in, 2) by trapping mixed-disulfide complexes of these proteins with their substrates (this will be done to define heretofore undetected substrates of these proteins), and 3) isolating suppressor mutations in strains missing many of these components. We anticipate that characterization of these suppressor mutations will reveal additional members of the protein family and provide new information on the function of known members. We will also determine how the thioredoxin family, members of which have highly conserved three-dimensional structures, often exhibit such different substrate specificities. Swap constructs and mutations that alter specificity differences between the thioredoxins 1 and 2 will shed light on this question. Selection for altered specificity mutations is based on the failure of thioredoxin 2 to reduce the enzyme methionine sulfoxide reductase. The genetic studies will be combined with structural information obtained in a collaborative effort with an X-ray crystallographer We will characterize in depth the mechanism of action of the protein DsbB. DsbB is a membrane protein that is required for the reoxidation of the thiol oxidase, DsbA, passing its electrons to quinines. We have dissected this process into several steps. A collection of DsbB mutants already isolated will be used to determine the role of different domains of the protein in this complex series of steps. Structural information on the protein obtained from both a collaborative NMR effort and genetic studies will be combined with this mutant analysis to understand the functioning of this protein. Studies on pathways of disulfide bond formation and reduction have already provided benefits for the enhanced production of medically important proteins such as antibodies and tissue plasminogen activators and this study should provide addition information for such efforts. Furthermore, these proteins play an important role in a host of cellular processes, both normal and patholological.