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Photochromic (i.e. reversibly photoswitchable) fluorescent proteins increasingly find applications as biomarkers for advanced bioimaging applications. From a mechanistic point of view, photochromicity usually stems from the reversible cis-trans photoisomerization of the chromophore. We demonstrated experimentally that cis-trans photoisomerization constitutes a very efficient deactivation pathway of isolated chromophores upon visible light excitation. Nonetheless, this intrinsic property is seldom displayed by chromophores in the folded protein structure. We found that the E222Q amino acid replacement restores efficient photochromicity in otherwise poorly switchable green fluorescent protein variants of different optical properties. Glutamic acid 222 is known to play a pivotal role in the inner proton wires that involve the GFP chromophore and the surrounding residues. Hence its substitution with an isosteric but non-ionizable residue presumably leads to a extensive rewiring of proton pathways around the chromophore, which has a deep effect also on the photochromic properties. In this work, we review and discuss the main photophysical properties of photochromic E222Q GFP mutants. Additionally we show, by means of flash-photolysis experiments, that chromophore cis to trans photoswitching involves a molecular mechanism where stereochemical isomerization and chromophore protonation occur in a coordinated way. Such a "concerted" mechanism is, in our opinion, at the basis of efficient photochromic behavior and might be activated by the E222Q mutation.

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.

The photophysical mechanism underlying the photochromic behavior of green fluorescent protein (GFP) mutants is investigated by means of preresonant Raman spectroscopy and model calculations. The studied molecules are reversibly switchable fluorophores that can be repeatedly converted between fluorescent and nonfluorescent states by irradiation and are attracting a broad interest for a number of new applications. Experimental results on chemically synthesized isolated chromophores are analyzed within a density functional theory approach that allows us to link the detected vibrational modes to specific ground-state configurations before and after photoconversion. These data are compared to results obtained for the case of complete folded proteins including the same chromophores. Our results highlight the impact of chromophore cis-trans isomerization and protonation state in the photophysics of these proteins and provide useful guidelines for novel mutant design.