Genetics of Bacterial Thiol Redox Proteins

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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.