The formation of DISULFIDE bonds is vital for the proper folding of most secreted proteins, including many of pharmacological importance. Great progress has been made in the last few years in understanding the mechanism of disulfide oxidation in vivo. We have recently succeeded in establishing the pathway of disulfide bond formation. This opens up the way for extensive biochemical and mechanistic analysis of the process of disulfide bond formation. DsbA acts as a direct donor of disulfides to secreted proteins. It then is reoxidized by DsbB. DsbB is the first enzyme known to use the oxidizing power of quinones to generate disulfides de novo; its catalytic activity is the primary source of disulfides in the cell. Analysis of DsbB's mechanism will give us insights as to how disulfides are created. DsbB binds and reduces quinones; DsbB also binds to DsbA but it oxidizes it, most likely via a thiol disulfide exchange reaction. I propose to study how DsbB interacts with quinones and DsbA. In a complementary approach, I will study how DsbA recognizes its two substrates: folding proteins and DsbB. The answer to these questions will give us insights into DsbA's chaperone action, protein-protein interactions as well as protein-quinone recognition. DsbA needs to very specifically recognize DsbB, but it also needs to be able to recognize a relatively wide spectrum of different folding proteins. We must clearly define and distinguish the residues in DsbA that are involved in unfolded protein and DsbB interaction. We must also clearly define and distinguish the residues in DsbB that are involved in quinone and DsbA interaction. To accomplish these goals, we will exploit powerful genetic selections, in vitro evolution, biochemical assays, structural studies and the use of inhibitory quinone analogues. To investigate the mechanism of DsbB action, we will trace the flow of redox equivalents from quinones through DsbB and then on to DsbA by measuring the rates at which various domains of DsbB react with each other, with DsbA and with quinones. Our work will help illuminate the mechanism of disulfide bond formation, a process vital for protein folding and reveal how protein folding factors are able to recognizes a relatively wide variety of partially folded proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Wehrle, Janna P
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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