Protein folding studies are directed towards the goal of achieving the capability of designing novel polypeptide sequences which will fold into desired shapes with medically useful activities. A potentially useful class of synthetic proteins would be small inhibitors of enzymes. Many natural inhibitors consist of globules with a protruding amino acid which resembles the enzyme's substrate, such that the inhibitors, competes for enzyme active sites in binding reactions. Enzyme inhibitors regulate many physiological processes such as complement fixation, blood- clotting, ovum fertilization, and cell-cell surface interactions. The objective of the proposed research is to develop strategies for directing formation of specific disulfides in a solution containing many different cysteines. The proposed research will study disulfide pairing between cysteine-containing peptides synthesized directly in the laboratory. Successful specific pairing would suggest synthetic sequences containing portions of the same peptides in a single chain, yielding the same pairing with a resultant specific topology. Different sequences could be designed for producing a collection of novel mainframes. These would serve as the basis for subsequent inhibitor production by addition of protruding amino acids at different locations on the mainframe. This project will test the hypothesis that the formation of specific bridges can be controlled by choosing appropriate positions for the cysteines in the polypeptide sequence and by surrounding the cysteines with appropriate electrostatically charged or sterically restrictive residues. Experiments will measure quantitative effects of such factors on the microscopic rate constants in disulfide exchange reactions. These rates will be used to suggest peptide mixtures where particular disulfides are predicted to predominate at equilibrium. The predictions will be evaluated experimentally by establishing disulfide interchange equilibria between peptides, quenching exchange with fluoroacetic acid or iodoacetamide, separating the products by HPLC and determining their relative concentrations. Data will be analyzed by Runga-Kutta numerical integration. Experiments also will evaluate whether sequences which exist as independently folded loops protruding from globular proteins are able to fold independently in solution, bringing cysteines at loop ends into close proximity.
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