Improper folding of proteins has been directly implicated in at least a dozen disease states including Alzheimer's and mad cow disease. Disulfide bonds are important enough for protein folding and stability that simple reduction of these bonds will often cause proteins to unfold. The formation of disulfide bridges is a catalyzed process. We found two catalysts to be involved, DsbA acts as the direct donor of DiSulfide Bonds to newly synthesized periplasmic proteins, DsbB acts to reoxidize DsbA. Our long term goal is to understand how these proteins act to catalyze protein folding. In this project we will seek to answer two basic questions: 1) Why is DsbA so powerful a protein oxidant? and 2) What are the catalytic properties of DsbB that allow it to specifically oxidize DsbA? Two distinct models have been proposed to explain the extreme oxidizing power of Dsba's active site disulfide relative to the related protein, thioredoxin. One model invokes disulfide strain, the other electrostatic interactions that affect the pKa of an active site cysteine. We have designed a multifaceted genetic, biophysical and structural approach that should clearly distinguish between these models. Strain and electrostatic interactions play important roles in the folding and catalytic function of many proteins. Clearly understanding the role of these forces in one model system should thus provide valuable information for understanding their role in other proteins as well. To analyze the redox and catalytic properties of DsbB we propose to use a workbox of tools very similar to those we have successfully used with DsbA. This straightforward characterization should tell us much about the way DsbB functions to reoxidize DsbA and may open the door to investigation of how disulfide bond formation is linked to cellular metabolism. Disulfide bond formation is one of the few covalent modifications that occurs in protein folding. This allows us to phrase our questions in simple biochemical terms. We feel that we now have the potential to understand the function of the DsbA-DsbB disulfide catalytic machine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057039-03
Application #
6151204
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1998-02-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
3
Fiscal Year
2000
Total Cost
$194,574
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Vertommen, Didier; Depuydt, Matthieu; Pan, Jonathan et al. (2008) The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner. Mol Microbiol 67:336-49
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Hiniker, Annie; Bardwell, James C A (2004) In vivo substrate specificity of periplasmic disulfide oxidoreductases. J Biol Chem 279:12967-73
Masip, Lluis; Pan, Jonathan L; Haldar, Suranjana et al. (2004) An engineered pathway for the formation of protein disulfide bonds. Science 303:1185-9

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