The objective of this project continues to be the use of NMR spectroscopy and nonradiative excitation energy transfer to determine the structures of intermediates (and thereby the interactions that lead to them) in the folding pathways of proteins. The primary aim is to solve structural problems related to biological function. Specifically, the aim is to solve the problem of how proteins fold into their native conformations. For this purpose, experimental techniques are used to provide an understanding of the internal interactions that stabilize native proteins in aqueous solution. Use is made of kinetic measurements, protein fractionation, peptide mapping, recombinant DNA procedures, NMR spectroscopy, and nonradiative excitation energy transfer to determine the pathways of folding of three members of the ribonuclease family - viz., four-disulfide bovine pancreatic ribonuclease A, and its homologs, three-disulfide bovine angiogenin and frog onconase. The experimental work will be concerned with the folding of the disulfide-reduced protein during the oxidation of its sulfhydryl groups and with the folding of the disulfide-intact protein from its denatured form. The oxidative folding experiments will be carried out to identify the kinetically relevant species (and structural features therein) that lead to one-, two-, etc. -disulfide intermediates and to the transitions between these intermediates along the folding pathways. The disulfide-intact experiments will provide important information to distinguish between rates of conformational changes and disulfide exchange, and information about structure formation as the protein folds. An understanding of the interactions in proteins is of potential applicability to the elucidation of the role of conformation in biological processes; e.g., the undesirable association of sickle-cell hemoglobin, or the induction of an oncogene product, whose properties involve a conformational change when only one amino acid residue in the sequence is changed. This supplemental application is a request for funds to purchase stopped-flow equipment. ? ?
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