9406275 Beckwith Many proteins exported from cells and many membrane proteins contain disulfide linkages between cysteines within their sequences. These disulfide bonds are often important both for the folding and the functioning of the protein. In 1991, it was found that bacteria express a protein, DsbA, that is required for efficient formation of disulfide bonds in proteins. Since then, it has been found that the yeast Saccharomyces cerevisiae expresses a similar activity. A major purpose of this project is to study the mechanism by which the DsbA protein catalyzes oxidation of pairs of cysteines in proteins. To this end, mutants are being isolated that are defective in DsbA activity, the mutant genes sequenced and in vivo studies carried out to define the defective step in the process. In addition, interaction and collaborations with biochemists and an X-ray crystallographer will help elucidate the basis of the defect. These studies should lead to a detailed picture of the structurefunction relationships for DsbA. To further understand the mechanism of DsbA action, experiments done in vivo are designed to indicate at what stage of protein synthesis and folding the DsbA protein acts on exported proteins to catalyze disulfide bond formation. In vivo and in vitro experiments will test whether DsbA can act on an already folded protein. In vivo experiments will analyze exported proteins during the process of membrane translocation to determine whether disulfide bonds can form before the proteins are fully translocated. A genetic selection has been devised which may allow the isolation of mutants of exported proteins that are unable to be acted on by systems that promote disulfide bond formation even though they retain the appropriate cysteines. beta-lactamase will be used as a model system since it has a single disulfide bond, is exported to the periplasm, is functional in the absence of its disulfide bond and has had its 3-dimensional structure determined to 1.8 Angst roms. These mutants may alter the folding, pathway for the protein or its secondary or tertiary structure. Analysis of the sequence and structure of the mutant proteins should reveal the important features that allow disulfide bond formation to take place. Experiments are also designed to detect other systems in the bacterial periplasm that catalyze disulfide bond formation. In the absence of the DsbA system, a background disulfide bond-forming activity is found. Mutants that both increase and decrease the activity of this background activity will be sought. In addition, genetic studies will be initiated to identify the system responsible for disulfide bond formation under conditions of anaerobic growth. These studies should lead to the identification of genes and gene products responsible for these other activities. %%% These studies have wide implications for basic and applied science. The results are relevant to questions of protein folding, one of the major new developing areas in biology. In addition, the formation of disulfide bonds in expressed foreign proteins in bacterial systems has presented many problems. The ability to manipulate the disulfide bond-forming systems should allow more ready solution to these expression problems. The range of important proteins that contain disulfide bonds includes peptide hormones, membrane receptors, bacterial toxins and immunoglobulins. ***
