Tryptophan phosphorescence has been used for over 20 years to study the conformation and flexibility of proteins under physiological conditions. The basis for this research has been the extraordinary sensitivity of the tryptophan triplet state to quenching by molecular collisions (protein flexibility). The triplet state photophysics of tryptophan and its analogs is inadequately understood; little is known of the actual physical mechanism by which collisions quench the triplet state and almost nothing is known about the triplet state photophysics and non-radiative decay of tryptophan analogs (which can be incorporated into proteins in vivo to generate spectrally enhanced proteins). This project will investigate the triplet state photophysics of trp and trp analogs at room temperature in glassy sugar matrixes and the effect of temperature and viscosity on these properties, additional studies will assay the effect of deuterium substitution on non-radiative decay of try ptophan to evaluate the influence of C-H stretch/bending modes on promoting the T to S transition. Measurements of quantum yields for fluorescence, triplet state formation, and phosphorescence will be combined with lifetime measurements to generate a complete photophysical characterization of the excited state photophysics of this important class of biological chromophores. Detailed comparison of the photophysical behavior in rigid (glassy sugar) and mobile (viscous solution) environments will allow evaluation of the quenching rate constant due to collisions for tryptophan and each of the analogs.
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