Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore

Abstract

Initiated by excited-state intramolecular proton transfer (ESIPT) reaction, an overall reaction cycle of 4-(2-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-HBDI), an analogue of the core chromophore of the green fluorescent protein (GFP), has been investigated. In contrast to the native GFP core, 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (p-HBDI), which requires hydrogen-bonding relay to accomplish proton transfer in vivo, o-HBDI possesses a seven-membered-ring intramolecular hydrogen bond and thus provides an ideal system for mimicking an intrinsic proton-transfer reaction. Upon excitation, ESIPT takes place in o-HBDI, resulting in a ∼600 nm proton-transfer tautomer emission. The o-HBDI tautomer emission, resolved by fluorescence upconversion, is comprised of an instantaneous rise to a few hundred femtosecond oscillation in the early relaxation stage. Frequency analysis derived from ultrashort pulse gives two low-frequency vibrations at 115 and 236 cm(-1), corresponding to skeletal deformation motions associated with the hydrogen bond. The results further conclude that ESIPT in o-HBDI is essentially triggered by low-frequency motions and may be barrierless along the reaction coordinate. Femtosecond UV/vis transient absorption spectra also provide supplementary evidence for the structural evolution during the reaction. In CH(3)CN, an instant rise of a 530 nm transient is resolved, which then undergoes 7.8 ps decay, accompanied by the growth of a rather long-lived 580 nm transient species. It is thus concluded that following ESIPT the cis-proton transfer isomer undergoes cis-trans-isomerization. The results of viscosity-dependent dynamics are in favor of the one-bond-flip mechanism, which is in contrast to the volume-conserving isomerization behavior for cis-stilbene and p-HBDI. Further confirmation is given by the picosecond-femtosecond transient IR absorption spectra, where several new and long-lived IR bands in the range of 1400-1500 cm(-1) are assigned to the phenyl in-plane breathing motions of the trans-proton transfer tautomer. Monitored by the nanosecond transient absorption, the 580 nm transient undergoes a ∼7.7 μs decay constant, accompanied by the growth of a new ∼500 nm band. The latter is assigned to a deprotonated tautomer species, which then undergoes the ground-state reverse proton recombination to the original o-HBDI in ∼50 μs, achieving an overall, reversible proton transfer cycle. This assignment is unambiguously supported by pump-probe laser induced fluorescence studies. On these standpoints, a comparison of photophysical properties among o-HBDI, p-HBDI, and wild-type GFP is discussed in detail.