The bacterial cytoplasm is normally maintained at a redox potential such that disulfide bonds do not form. Outside the inner membrane the redox potential often is such that disulfide bonds in proteins do form, and evolution likely has utilized these bridges to add stability to proteins. Two problems are generated by the utilization of such bonds. First, the rate of oxidation of cysteine pairs to form a disulfide bond can be slow, and second, interchange of disulfide bonds is often necessary to shuffle these bonds until the correct set has been made in a protein. E. coli apparently possesses a pathway for the formation and isomerization of disulfide bonds of proteins secreted into and through the periplasmic space. The work proposed is focussed on two objectives, how are proteins that contain multiple disulfide bonds assembled, and how can E. coli be engineered to optimize the assembly of such proteins? The first objective will be studied in Dr. Beckwith's laboratory in the Harvard Medical School, and the second will be carried out as a consortium/contract by Dr. George Georgiou at the University of Texas, Austin. The stated goals are to characterize the pathway that maintains disulfide bond isomerase, DsbC, in a reduced state, to isolate mutants that affect this pathway, and to seek additional genes and proteins that play a role in the pathway. Genetic experiments toward these ends will be performed in Beckwith's laboratory, and biochemical experiments in Georgiou's laboratory. The following disulfide proteins will be used in the studies, endonuclease I, bovine pancreatic trypsin inhibitor, and tissue plasminogen activator. Structure-function studies, meaning membrane topology, will be performed on DsbD and the interactions of DsbC with cytoplasmic and extracytoplasmic components will be examined. Genes whose overexpressed protein products will suppress DsbC mutants will be sought.
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