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Toward Understanding Tryptophan Fluorescence in Proteins
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1998
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The study presents a general approach to dissect tryptophan photophysics to elucidate how amino‑acid functional groups affect its fluorescence. The authors employed steady‑state and time‑resolved fluorescence, H‑D exchange, and transient absorption to quantify nonradiative deactivation pathways, including intersystem crossing, solvent quenching, and excited‑state proton and electron transfer. Eight amino‑acid side chains were shown to quench tryptophan fluorescence over a 100‑fold range, with lysine and tyrosine quenching via excited‑state proton transfer and glutamine, asparagine, glutamic, aspartic, cysteine, and histidine quenching via excited‑state electron transfer, providing a framework for interpreting fluorescence data in proteins.
A general approach to dissecting the complex photophysics of tryptophan is presented and used to elucidate the effects of amino acid functional groups on tryptophan fluorescence. We have definitively identified the amino acid side chains that quench tryptophan fluorescence and delineated 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 used 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 rates. Eight amino acid side chains representing six functional groups quench 3-methylindole fluorescence with a 100-fold range in quenching rate constant. Lysine and tyrosine side chains quench by excited-state proton transfer; glutamine, asparagine, glutamic and aspartic acid, cysteine, and histidine side chains quench by excited-state electron transfer. These studies provide a framework for deriving detailed structural and dynamical information from tryptophan fluorescence intensity and lifetime data in peptides and proteins.