Disulfide bond formation is an integral part of the expression of numerous extracellular proteins including receptors, enzymes, and peptide hormones. A fundamental understanding of disulfide bond formation will ultimately be essential to the design of any disulfide-bonded protein or peptide targeted to the extracellular environment by gene therapy expression systems. Shortly after translation on the endoplasmic reticulum, proteins containing disulfide bonds must correctly fold and form specific sets of disulfide bonds. This process is very rapid in vivo, and is likely to be catalyzed by an enzyme--protein disulfide isomerase. The long-term goal of this research is to understand the mechanism of correct disulfide bond formation, to describe the thermodynamic and kinetic constraints on the intracellular compartment where disulfide bond formation occurs, and to investigate mechanisms by which disulfide bond formation can be catalyzed. Simple predictions of the rate of oxidation and reduction of dithiols and disulfides suggest that the effectiveness of any catalyst for protein oxidation will depend on the relationship between the redox status of the environment and the oxidation potentials of the protein and any catalyst. Immediate experimental goals involve determining the efficiency of catalyzed and uncatalyzed protein oxidation and reduction as a function of the oxidation potential and redox status of the redox buffer. A series of hydrophilic peptides containing two cysteine residues located 1 to 9 residues apart will be synthesized to provide a spectrum of redox buffers of different oxidation potentials. These peptide redox buffers will be used to set the redox poise of the medium in which protein disulfide bond formation occurs. The formation and cleavage of disulfide bonds in several well-characterized proteins (bovine trypsin inhibitor, ribonuclease A, and lysozyme) will be studied in the presence and absence of protein disulfide isomerase in these dithiol/disulfide peptide redox buffers. The oxidation potential of protein disulfide isomerase will be determined. The achievement of these experimental objectives will lead to an enhanced understanding of the complex mechanisms by which protein disulfide bonds are formed during the expression of disulfide-bonded proteins and how these processes are catalyzed.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Baylor College of Medicine
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