Ultrafast Decay of Trp in Biological Macromolecules

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Femtosecond (<300fs fwhm) measurements of fluorescence decay and quenching of Tryptophan (Trp) were performed in a variety of proteins, including GB1, Thioredoxin (both wild type from two species and a human Trx mutant with a single Trp), Cyanovirin and Interleukin-1beta using an upconversion spectrophotofluorometer combined with a time correlated single photon counting apparatus to span the ∼200fs to 20ns time scale. Trp is subject both to ultrafast quenching in proteins and spectral energy loss coupled to nearby water dynamics. All fluorescence transients of tryptophan in proteins reveal complex, i.e. multiexponential behavior. In addition to a “bulk water” relaxation (∼2 ps), a 50 ps fluorescence decay was found in single-Trp thioredoxin which matched the component we had found previously in two-Trp Anabaena and E. coli thioredoxins . In fact, a sub-100ps component is consistently found in all but one of these proteins with positive amplitudes even at longer wavelengths (e.g., 390nm). The exception is GB1, a protein which Toptygin and Brand previously found carried a negative preexponential term near 390nm. Since the lifetime associated with that negative was 65ps, it was just within the edge of TCSPC detection. The more prevalent positive amplitude DAS (decay-associated spectra) we see in the other proteins on these timescales are indicative of ultrafast quenching processes depleting the partly relaxed singlet state. Candidate mechanisms include ET to nearby acceptors and/or collisional quenching. This is similar to our prior observation (J. Am. Chem. Soc., 2006, 128, 1214) that ultrafast quenching, not desorbing water, dominates the time-resolved emission spectra (TRES) of monellin. We will discuss the −/+ data signatures for both water relaxation and fast quenching, including simulations to lay out the circumstances where a fast relaxation accompanied by a direct radiative rate reduction might mask the negative term.