NSF Award
9306367
9306367 Roder The main objective of the proposed work is to gather kinetic and structural information on the initial stages of protein folding. The time resolution of kinetic folding studies will be extended into the sub-millisecond time range by using laser flash photolysis and improved rapid mixing techniques to initiate the folding reaction. The resulting conformational changes will be monitored by transient spectroscopy in the visible and near-UV region, as well as steady-state fluorescence and picosecond time-resolved fluorescence. These techniques will be applied to cytochrome c and variants prepared by site-directed mutagenesis. By taking advantage of the redox and ligand binding properties of the covalently attached heme group, it is possible to initiate the folding reaction of cytochrome c by photoreduction, or by photodissociation of a carbon monoxide ligand bound to the reduced heme iron in the unfolded protein. The collapse of the polypeptide chain will be monitored by time-resolved fluorescence in conjunction with rapid mixing techniques, initially using continuous-flow and later a jet mixing device for measurements on a sub-millisecond time scale. %%% The question of how a polypeptide chain can spontaneously fold into a biologically active protein is of fundamental and practical importance throughout molecular biology, but we are only just beginning to unravel some of the principles of this remarkable reaction. Because of the limited time resolution of conventional kinetic techniques, there are at present no direct experimental observations on protein folding events on the sub- millisecond time scale. Yet, these rapid structural events are key to understanding how protein folding is initiated, which is the focus of numerous theoretical efforts to solve the protein folding problem. Kinetic studies involving novel optical triggering and fast mixing methods will provide unique insight into chain collapse and secondary formation during early stages of protein folding. ***
