This proposal describes studies of thiol redox proteins of E. coli in terms of their function and as ways of exploring features of the process of protein translocation across the cytoplasmic membrane. The specific objectives are: 1. to use mutant analysis to examine the role of protein folding in the process of translocation of the normally cytoplasmic thioredoxin into the E. coli periplasm; 2. to use mutant analysis and biochemical studies to determine the relationship between the structure and function of two cell envelope protein, DsbA and DsbB, required for disulfide bond formation in proteins; 3. to determine, for those cell proteins that contain disulfide bonds, when during the protein translocation process the DsbA acts to oxidize cysteines; to analyze the role of different pairs of cysteines in the function of the unusual newly discovered thioredoxin, TrxC; 5. to determine the nature of the specific interactions between thiol redox proteins such as thioredoxin and its substrates. The major component of this work involves protein analysis, although the structure-function studies with DsbA and DsbB include a collaborative effort with laboratories analyzing structural and biochemical features of these proteins. These studies should be useful particularly for engineering bacterial strains that more efficiently produce proteins of medical utility including hormones and antibodies. Strains isolated under this project in the past have been used for such useful purposes.
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