Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer.

Steady-state emission, femtosecond pump-probe spectroscopy, and time-correlated single-photon counting (TCSPC) measurements were used to study the photophysics and the excited-state proton transfer (ESPT) reactions in the green fluorescent protein (GFP) variant S65T/H148D at three pH values: 6.0, 7.9, and 9.5. Selective mutation of GFP caused a dramatic change in the steady-state and excited-state behavior as compared to the wild-type GFP (wt-GFP) studied earlier. An excitation wavelength dependence of the quantum yield of the strong emission band at 510 nm (I* band) indicates the competition between adiabatic and non-adiabatic excited-state proton-transfer reactions. Pump-probe measurements show that the signal buildup probed at 510 nm is biphasic, where 0.8 of the signal amplitude is ultrashort, <150 fs, and 0.2 of the signal decreases with a approximately 10 ps time constant. This effect is a summary result of adiabatic ESPT to the carboxylate group of Asp148 and nonradiative processes. When compared with the luminescence of wt-GFP, the steady-state intensity at 450 nm is lower by a factor of about 10. This very weak emission at 450 nm has a nonexponential decay. It is relatively pH insensitive and, at about 25 ps, is almost twice as long as in wt-GFP. The former exhibits a surprisingly small kinetic deuterium isotope effect (KDIE), compared with the KDIE of about 5 for wt-GFP. Such weak proton dependence may indicate that this emission comes from the species not directly involved in the ESPT. In contrast, pH and H/D isotope dependence of the intense nonexponential luminescence decay of the S65T/H148D deprotonated form measured at 510 nm may result from an isomerization-induced deactivation that is accompanied by the proton recombination quenching. The data are complementary to the femtosecond time-resolved emission data obtained by ultrafast fluorescence up-conversion spectroscopy, found in the preceding paper (Shi et al.). The spectroscopic results are discussed on the basis of the detailed X-ray structure of the mutant published in the preceding paper (Shu et al.).