TIME-RESOLVED SPECTROSCOPY OF WILD-TYPE AND MUTANT GREEN FLUORESCENT PROTEINS REVEALS EXCITED-STATE DEPROTONATION CONSISTENT WITH FLUOROPHORE-PROTEIN INTERACTIONS

Recently steady-state and picosecond time-resolved absorption and fluo rescence spectroscopy on the Green Fluorescent Protein (GFP) have been interpreted by a mechanism where the key process is an excited state deprotonation of the chromophore (M. Chattoraij, B.A. King, G.U. Bubli tz and S.G. Boxer, Proc. Natl. Acad. Sci. USA, 93 (1996) 8362-8367). S uch a conclusion was borne out by the mirror image of the picosecond d ecay of the protonated species RH in the blue and the concomitant pic osecond rise of the green fluorescence of the deprotonated fluorophore R(-) as well as the significant slowing of both kinetic features upo n deuteration. We report similar experiments confirming this mechanism . The results of ultrafast spectroscopy on wild-type GFP together with two important mutants combined with the recent crystal structures are shown to shed more light on the interplay between absorption and emis sion phenomena in GFP. Beyond some differences with previous results p ertaining, for instance, to the assignment of vibronic progressions in absorption spectra and the temperature dependence of excited state de protonation, several new features have been identified. These concern the deprotonated ground state R(-) in equilibrium as well as the excit ed state RH. In particular, we have studied the distributed fluoresce nce kinetics in the time and frequency domain, excited state absorptio n features observed in femtosecond time-resolution, and the dependence of excited state proton transfer kinetics on the aggregational state of the protein.