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

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...

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Published in:Journal of the American Chemical Society 2011-03, Vol.133 (9), p.2932-2943
Main Authors: Hsieh, Cheng-Chih, Chou, Pi-Tai, Shih, Chun-Wei, Chuang, Wei-Ti, Chung, Min-Wen, Lee, Junghwa, Joo, Taiha
Format: Article
Language:English
Subjects:
Biomimetic Materials - chemistry
Green Fluorescent Proteins - chemistry
Hydrogen Bonding
Imidazoles - chemistry
Kinetics
Protons
Spectrometry, Fluorescence
Spectrophotometry
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Summary: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 CH3CN, 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 s
ISSN:0002-7863
1520-5126
DOI:10.1021/ja107945m