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

Author

Listed:
  • 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
    Institut Laue Langevin)

  • 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
    Laboratoire Chimie et Biologie des Métaux, BIG, CEA-Grenoble)

  • 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)

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.

Suggested Citation

  • 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 & Fran, 2020. "Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-14537-0
    DOI: 10.1038/s41467-020-14537-0
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