James Bardwell

One of the fundamental unsolved problems in biology is how proteins attain their proper three-dimensional conformation. Protein folding catalysts are vital in assisting proteins in this process. My laboratory is interested in understanding the molecular mechanism of these folding helpers. Understanding how the protein folding process is assisted is important to understand not just the vital protein folding process itself, but also the numerous pathologies, like Alzheimer's and cystic fibrosis, that result from defective protein folding. We study the disulfide bond formation step in protein folding. We found that this is a catalyzed process, and have shown how disulfide bond formation is linked to cellular metabolism. One indication of how vital disulfide bonds are for protein folding and stability is the fact that their reduction will often cause proteins to unfold. We made the unexpected finding that disulfide bond formation is catalyzed. We developed a disulfide indicator strain and used it to select for mutants severely defective in disulfide bond formation. We discovered the DsbA protein and showed that the active site of DsbA is itself a disulfide bond that is reduced catalytically using disulfide bond formation on folding proteins. We also found a second protein DsbB, which acts to reoxidize DsbA. We have recently succeeded in the in vitro reconstitution of the complete disulfide bond catalysis system. We determined where the oxidative power for protein folding originates and have shown that disulfide bond formation is linked to electron transport. DsbB transfers pairs of electrons on to ubiquinone, which then donates them to cytochrome oxidases, which reduce oxygen. These findings open up an experimentally approachable system to study the catalysis of an important step in the protein folding process. Since disulfide bond formation is one of the few covalent modifications that occurs in protein folding we are in the unusual and advantageous position of being able to phrase our questions about the catalysis of a protein folding reaction in clear biochemical terms. Our findings suggest that catalysts may be required for disulfide formation in all organisms, a fact that is now generally accepted. Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides, including many proteins of pharmacological importance. Great progress has been made in the last few years in understanding the mechanism of disulfide oxidation in vivo, but the mechanism of isomerization is much less clear. We have shown how the oxidative and isomerase pathways are kept separate in the cell; we have used a combination of genetics, biochemistry and in vitro protein design to convert the isomerase, DsbC into a net donor of disulfide bonds.

Research Areas(s)

• Protein Folding, Heat Shock Proteins