One of the major unsolved problems of molecular biophysics involves determining how proteins fold. Steady-state fluorescence changes of intrinsic tryptophan fluorescence have been utilized to monitor folding transitions in proteins for many years. From these studies, only kinetic arguments concerning the mechanism of folding transitions could be made. In this proposal, a new class of fluorescence experiments are introduced: stooped-flow time- resolved fluorescence. In these studies, a high repetition picosecond laser is utilized for excitation, and the fluorescence emission is detected by sixteen independent time-resolved single photon counting channels. In this manner, complete time-resolved fluorescence decay can be obtained in as little as 10 milliseconds. The addition information content of the time-resolved experiments allows stopped-flow studies to examine not just the kinetics of a transition but also the mechanism. Studies are performed on the single tryptophan containing Staphylococcal Nuclease and site- specific mutants. Changes in the intrinsic tryptophan fluorescence in this protein was first used by Anfinsen's group in 1971 to unequivocally demonstrate the existence of kinetic intermediates in the refolding of proteins. However, no insight into the physical nature of the intermediate form(s) of this protein have ever been obtained. From examination of the millisecond changes in lifetimes and rotational correlation times of nuclease during refolding, the time-course of localized and native-like structure formation are observed, as well as the solvent exposure history of the single tryptophan residue. A series of site-specific mutants have been constructed, specifically designed for performing energy transfer experiments between the single tryptophan and acceptor groups located throughout the structure of nuclease. From examination of these mutants millisecond motion pictures of the refolding transition are obtained. Although Staphylococcal Nuclease and its site-specific mutants are targeted for investigation, the methodologies developed in this proposal will be beneficial for all studies which utilize the kinetics of changes in steady-state fluorescence to monitor biological transitions (e.g., ligand binding, protein association reactions, protein:DNA interactions, etc.).
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