NOVEL ROLE FOR CYSTEINE IN PROTEIN FOLDING ASSEMBLY

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Efficient production of proteins from recombinant genes is a critical issue in the pharmaceutical and biotechnology industries, and in biochemical and biomedical research. However, high level expression of cloned genes does not always lead to a proportional increase in biologically active product. Often, misfolding leads to proteolytic degradation or aggregation. The complex nature of protein expression in both eukaryotic and prokaryotic systems points to the need for a more complete understanding of the factors which determine both intracellular and extracellular polypeptide chain aggregation. To study amino acid determinants of folding and misfolding, P22 bacteriophage tailspike protein will be used as a model system. The folding pathway of P22 tailspike is well characterized and the recently published crystal structure reveals several novel structural motifs. Recent results suggest that one or more cysteine residues present in the interdigitated B sheet region are activated during folding and assembly of the trimer. The specific goals of this proposal are to characterize the role of the active cysteine residues in folding and trimer assembly using traditional biochemical techniques and site-directed mutagenesis, and by altering the redox environment. Our motivation is to provide an understanding of the determinants of folding, misfolding, assembly, and aggregation which will result in an improved ability to suppress or control nonproductive processes in folding.