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

Joyce Woodhouse, Gabriela Nass Kovacs, Nicolas Coquelle, Lucas M. Uriarte, Virgile Adam, Thomas R. M. Barends, Martin Byrdin, Eugenio Mora, R. Bruce Doak, Mikolaj Feliks, Martin Field, Franck Fieschi, Virginia Guillon, Stefan Jakobs, Yasumasa Joti, Pauline Macheboeuf, Koji Motomura, Karol Nass, Shigeki Owada, Christopher M. Roome, Cyril Ruckebusch, Giorgio Schirò, Robert L. Shoeman, Michel Thepaut, Tadashi Togashi, Kensuke Tono, Makina Yabashi, Marco Cammarata, Lutz Foucar, Dominique Bourgeois, Michel Sliwa (), Jacques-Philippe Colletier, Ilme Schlichting () and Martin Weik ()
Additional contact information
Joyce Woodhouse: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Gabriela Nass Kovacs: Max-Planck-Institut für medizinische Forschung
Nicolas Coquelle: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Lucas M. Uriarte: Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectrochimie Infrarouge et Raman
Virgile Adam: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Thomas R. M. Barends: Max-Planck-Institut für medizinische Forschung
Martin Byrdin: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Eugenio Mora: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
R. Bruce Doak: Max-Planck-Institut für medizinische Forschung
Mikolaj Feliks: University of Southern California
Martin Field: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Franck Fieschi: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Virginia Guillon: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Stefan Jakobs: Max Planck Institute for Biophysical Chemistry
Yasumasa Joti: Japan Synchrotron Radiation Research Institute
Pauline Macheboeuf: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Koji Motomura: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University
Karol Nass: Max-Planck-Institut für medizinische Forschung
Shigeki Owada: RIKEN SPring-8 Center
Christopher M. Roome: Max-Planck-Institut für medizinische Forschung
Cyril Ruckebusch: Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectrochimie Infrarouge et Raman
Giorgio Schirò: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Robert L. Shoeman: Max-Planck-Institut für medizinische Forschung
Michel Thepaut: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Tadashi Togashi: Japan Synchrotron Radiation Research Institute
Kensuke Tono: Japan Synchrotron Radiation Research Institute
Makina Yabashi: RIKEN SPring-8 Center
Marco Cammarata: UMR UR1-CNRS 6251, University of Rennes 1
Lutz Foucar: Max-Planck-Institut für medizinische Forschung
Dominique Bourgeois: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Michel Sliwa: Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectrochimie Infrarouge et Raman
Jacques-Philippe Colletier: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale
Ilme Schlichting: Max-Planck-Institut für medizinische Forschung
Martin Weik: Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-14537-0

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