期刊
NATURE COMMUNICATIONS
卷 11, 期 1, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41467-020-14537-0
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资金
- CEA
- CNRS (PEPS SASLELX)
- Lille University
- Max Planck Society
- FRISBI within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-INSB-05-02]
- GRAL within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-LABX-49-01]
- CNRS
- UGA
- Chevreul Institute
- Ministere de l'Enseignement Superieur et de la Recherche
- Region Nord-Pas de Calais
- FEDER
- E.U. under the program FP7PEOPLE-2011-ITN NanoMem [317079]
- CEA through the IRTELIS PhD program
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 mu s 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.
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