TOWARD UNDERSTANDING TRYPTOPHAN FLUORESCENCE IN PROTEINS

A general approach to dissecting the complex photophysics of tryptopha n is presented and used to elucidate the effects of amino acid functio nal groups on tryptophan fluorescence, We have definitively identified the amino acid side chains that quench tryptophan fluorescence and de lineated the respective quenching mechanisms in a simple model system. Steady-state and time-resolved fluorescence techniques? photochemical H-D exchange experiments, and transient absorption techniques were us ed to measure individual contributions to the total nonradiative rate for deactivation of the excited state, including intersystem crossing, solvent quenching, and excited-state proton and electron transfer rat es. Eight amino acid side chains representing six functional groups qu ench 3-methylindole fluorescence with a 100-fold range in quenching ra te constant. Lysine and tyrosine side chains quench by excited-state p roton transfer; glutamine, asparagine, glutamic and aspartic acid, cys teine, and histidine side chains quench by excited-state electron tran sfer. These studies provide a framework for deriving detailed structur al and dynamical information from tryptophan fluorescence intensity an d lifetime data in peptides and proteins.