FUNCTIONAL ANALYSIS OF A PROTEIN FOLDING CATALYST

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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.