Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.
Photobleaching, the irreversible photodestruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between nonfluorescent and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue. We investigated the photofatigue mechanisms in the protein IrisFP using combined X-ray crystallography, optical in crystallo spectroscopy, mass spectrometry and modeling approaches. At laser-light intensities typical of conventional wide-field fluorescence microscopy, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. At laser-light intensities typical of localization-based nanoscopy (>0.1 kW/cm(2)), a completely different, oxygen-independent photobleaching pathway was found to take place. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp(2)-to-sp(3) hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. This two-regime photobleaching mechanism is likely to be a common feature in RSFPs from Anthozoan species, which typically share high structural and sequence identity with IrisFP. In addition, our results suggest that, when such FPs are used, the illumination conditions employed in localization-based super-resolution microscopy might generate less cytotoxicity than those of standard wide-field microscopy at constant absorbed light-dose. Finally, our data will facilitate the rational design of FPs displaying enhanced photoresistance.
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