Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration

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Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration, J. Phys. Chem. Lett. 2017, 8, 1489−1495

 

Reversibly photoswitchable fluorescent proteins (RSFPs) are green fluorescent protein (GFP) homologues that can be switched back and forth between a fluorescent (ON) state and a nonfluorescent (OFF) state by irradiation at two wavelengths. Over the past decade, they have become essential tools for super-resolution microscopy and other advanced fluorescence imaging techniques. X-ray crystallography studies of the ON and OFF states of several RSFPs revealed that in most cases, photoswitching is based on cis/trans isomerization of the chromophore coupled with proton transfer. The photoswitching pathways of this class of RSFPs, the prototype of which is Dronpa, have been further investigated by timeresolved spectroscopy with visible and infrared (IR) probes, quantum chemistry, and low-temperature X-ray crystallography in order to establish the sequence in time of the two events, their characteristic time scales, and the detailed mechanisms. Although questions remain, a clear picture of chromophore transformations involved in the OFF →  ON photoswitching of Dronpa has recently emerged: the first step consists of picosecond trans →  cis isomerization of the chromophore in the excited state, while proton transfer follows much later in the ground state, on the microsecond time scale.

 

 

In conclusion, we studied the photoswitching dynamics of Dreiklang for the first time by time-resolved spectroscopy. We found that the ON →  OFF photoconversion, which ultimately results in chromophore hydration, is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state, with a time constant of 100 fs. This primary reaction is accompanied by coherent oscillations that we assign to the coupling of the reactive motion with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network.

 

Consultez le communiqué de presse associé à cet article : Comprendre le comportement d’un chromophore fluorescent !

 

 

Résumé: 

J. Phys. Chem. Lett. 2017, 8, 1489−1495

 

Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the fi rst timeresolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond− nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.

 

 

 

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Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration

 

Fabien Lacombat, Pascal Plaza, Marie-Aude Plamont, and Agathe Espagne

 

J. Phys. Chem. Lett. 2017, 8, 1489−1495

 

DOI: 10.1021/acs.jpclett.7b00348