Abstract
Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump–probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
Highlights
Switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging
Phototransformable fluorescent proteins (PTFPs) are invaluable tools for advanced fluorescence microscopy, serving as genetically encoded markers that change emission color or intensity when irradiated with visible light at specific wavelengths[1,2]
The first investigation of Dronpa by ultrafast optical spectroscopy suggested that the deprotonation of the trans-phenol occurs in the excited state on the ps timescale via an excited-state proton transfer (ESPT) mechanism[10]
Summary
Switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Picosecond time-resolved infrared (TR-IR) spectroscopy on Dronpa[11] and its fast-switching M159T mutant[12] indicated that isomerization occurs during picosecond excited-state decay This has been suggested by femtosecond UV–visible transient anisotropy absorption spectroscopy, which attributed the first photoproduct to a cis-protonated chromophore[13]. TR-IR study on the Dronpa-M159T mutant advocated that both isomerization and deprotonation are ground-state processes, attributing the ps spectroscopic changes in the excited state to protein conformational changes priming the chromophore for switching[14]. This view was corroborated by a follow-up TR-IR study on Dronpa-M159T involving isotope labeling[15]. Whereas both hydrozoan and anthozoan RSFPs have the same overall protein fold and chromophore (4-(p-hydroxybenzylidene)−5-
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