The long term goal of this project is to develop a quantitative understanding of the roles of individual amino acid residues and interactions in directing the folding of globular proteins. Mutational analysis, in conjunction with biochemical experiments and high- resolution NMR spectroscopy, will be used to study intermediates in the disulfide-coupled refolding of two small proteins, bovine pancreatic trypsin inhibitor (BPTI) and an omega-conotoxin. The major disulfide-bonded intermediates in the refolding of BPTI are presently among the best characterized for any protein. In order to determine what interactions and structural features favor the formation of the different intermediates, and how these contributions change as folding proceeds, the effects of sequence changes on the stabilities and kinetic roles of the intermediates will be measured. The modifications to be studied are designed either to eliminate side-chain hydrogen bonds, force changes to the native backbone conformation, or alter the covalent connectivity of the backbone. To obtain a more detailed understanding of how individual residues influence polypeptide conformation and dynamics, NMR spectroscopy will be used to examine the effects of amino acid replacements on native BPTI and an intermediate that lacks one of the three disulfides. The omega-conotoxins are unusually small disulfide-bonded proteins that function as antagonists of voltage-gated Ca2+ channels. In spite of their small size, 25-30 residues, they are able to form their three disulfides and fold into well-defined three-dimensional structures. To test for the presence and roles of stabilizing interactions early in folding, the relative stabilities of all of the possible one-disulfide intermediates will be determined, and the influence of each of the native disulfides on forming a second native disulfide will be measured. NMR spectroscopy will be used to study the flexibility and conformations of the three species containing two of the native disulfides, and mutational analysis will be used to asses the contributions of selected non-Cys residues to the stability of the folded conformation. The results of this project will contribute to the fundamental knowledge necessary to realize the health care potentials offered by the revolutionary developments in genetic analysis and manipulation. Possible applications of these results include the design of proteins with enhanced stabilities or novel activities and improved understanding of how naturally occurring mutations lead to disease states by preventing protein folding or altering the structure or dynamics of folded proteins.
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