Disulfide Bond Formation: Mechanisms for Isomerization and Novel Pathways

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Project Summary: Proteins containing disulfide bonds are found in all organisms. These bonds are important for the folding of such proteins into its tertiary structure. Organisms have evolved protein disulfide isomerases which insure that the correct disulfide bonds are found in the final product. In the bacterium E. coli, the DsbC protein corrects incorrectly formed disulfide bonds. DsbC is regenerated as an active enzyme by the membrane protein DsbD. We will study the mechanisms of action of DsbC and DsbD using genetic, biochemical and structural approaches. Isolation and characterization of mutants of dsbC, genetic engineering of it and studies on its interaction with misoxidized substrates should yield information on the amino acids required for its functioning, structural features important for it to work properly and what aspects of disulfide bond formation necessitate the existence of such isomerases. DsbD transfers electrons across the cytoplasmic membrane from thioredoxin to the periplasmic DsbC by a disulfide bond reduction cascade. Isolation and characterization of mutants of the dsbD gene combined with biochemical studies on mutant proteins and structural analysis should illuminate the unusual electron transfer mechanism involving the membrane-embedded domain of DsbD. We will develop strains altered either in their cytoplasmic redox state or in components of the periplasm that influence disulfide bond formation. Strains obtained provide means of producing higher yields of disulfide-bonded proteins than normal bacterial strains. Medically important proteins- e.g. insulin, human growth hormone and antibodies- contain disulfide bonds essential for their activity. Understanding features of disulfide bond formation may lead to insights into the disruption of these proteins' activities in cases of malfunction. Understanding mechanisms involved in disulfide bond formation can enhance the ability to efficiently produce some of these proteins for medical purposes. Relevance: Many proteins important to human growth and physiology contain chemical bonds between the sulfurs of two cysteines. These disulfide-bonded proteins include a large number of peptide hormones (e.g. insulin) and immunoglobulins (antibodies). Understanding how disulfide bonds are formed correctly gives information allowing efficient production of these proteins for medical purposes.