The goal of this research is to understand the influence of the local environment on the fluorescence decay of tryptophan residues in proteins. The fluorescence of indole, 3-methyl indole and tryptophan will be studied with the aid of pulse nanosecond fluorescence methods. Fluorescence decay data with excitation at various wavelengths will be obtained as a function of viscosity and solvent polarity. Experiments will be done to photoselect subpopulations of differentially solvated fluorophores. The data will be represented as fluorescence decay associated spectra (DAS) and time-resolved emission spectra (TRES). Theoretical studies will be done in order to predict the influence of excitation wavelength and environmental conditions such as viscosity, temperature and polarity on excited-state solvation processes. Similar studies will be done with several single tryptophan containing proteins including mutant forms of Enzyme I of the PTS, the galactose repressor, myelin basic protein and the ultrathioborax homeodomain (UBX). These experiments are designed to provide information about the mutual interaction between tryptophan residues in proteins and the polar solvent environment to provide more detailed information about the relation between protein structure and fluorescence and to explain the changes observed when proteins exhibit conformational changes or interact with other molecules.
The functions of proteins include catalytic activity and regulation. These events involve conformational changes observable by tryptophan fluorescence. A clear understanding of the origin of the sensitivity of tryptophan fluorescence to conformational changes of proteins remains elusive. The aim of these studies is to utilize nanosecond time-resolved fluorescence methods to understand the influence of excited-state solvation and charges on the fluorescence of tryptophan in proteins. This work will enhance the application of fluorescence spectroscopy to studies of function and structure of proteins.
