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Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

2020; Nature Portfolio; Volume: 11; Issue: 1 Linguagem: Inglês

10.1038/s41467-020-14537-0

2041-1723

Joyce Woodhouse, Gabriela Nass Kovács, Nicolas Coquelle, Lucas M. Uriarte, Virgile Adam, Thomas R. M. Barends, Martin Byrdin, E. De la Mora, R. Bruce Doak, Mikołaj Feliks, Martin J. Field, Franck Fieschi, Virginia Guillon, Stefan Jakobs, Yasumasa Joti, Pauline Machebœuf, Koji Motomura, Karol Nass, Shigeki Owada, C.M. Roome, Cyril Ruckebusch, Giorgio Schirò, Robert L. Shoeman, Michel Thépaut, Tadashi Togashi, Kensuke Tono, Makina Yabashi, Marco Cammarata, L. Foucar, Dominique Bourgeois, Michel Sliwa, Jacques‐Philippe Colletier, Ilme Schlichting, Martin H. Weik,

Advanced Electron Microscopy Techniques and Applications

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.