Steady-state and nanosecond time-resolved fluorescence methods will be used to investigate the dynamic and static structures of proteins and macromolecular assemblies. Complex fluorescence decay will be investigated so as to obtain new information about biological. systems. Use will be made of overdetermination together with global analysis methods so as to maximize the information that is obtainable. Work will be done to further develop and provide additional evidence for a theory of fluorescence depolarization and decay that takes orientational dependence into account. The theory predicts non- exponential emission intensity decay and emission anisotropy decay different than the one expected in optically-isotropic systems. Resonance energy transfer measured by both steady-state and timeresolved methods will be used to study several systems including a series of triantennary glycopeptides, mutant forms of staphylococcal nuclease and enzymes of the phosophotransferase system. Experiments will continue aimed at better understanding the role of Zn and SH residues in maintaining the tertiary and quaternary structure of liver alcohol dehydrogenase. Studies will be continued on the monomer/dimer transition of Enzyme I of the PTS and on the interactions between the component proteins of the PTS including Enzyme I, HPr, and Enzyme III-Glc. These studies will be done both in solution and with the proteins, together with phospholipid vesicles. These studies will be done both with the native forms and with mutant forms of the PTS proteins. An advantage of fluorescence spectroscopy is that the measurements are applicable to very heterogeneous systems and these methods can be used to study molecular interactions in the living cell. This cannot be done with classical hydrodynamic techniques. The nanosecond fluorometer will be combined with the fluorescence microscope. The nanosecond microspectrofluorometer will initially be used to study intracellular ion fluctuations (mainly Ca 2+) in A431 cells.
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