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

Woodhouse, Joyce; Nass Kovács, Gabriela; Coquelle, Nicolas; Uriarte, Lucas M.; Adam, Virgile; Barends, Thomas; Byrdin, Martin; de la Mora, Eugenio; Doak, Bruce; Feliks, Mikolaj; Field, Martin; Fieschi, Franck; Guillon, Virginia; Jakobs, Stefan; Joti, Yasumasa; Macheboeuf, Pauline; Motomura, Koji; Nass, Karol; Owada, Shigeki; Roome, Christopher M.; Ruckebusch, Cyril; Schirò, Giorgio; Shoeman, Robert L.; Thepaut, Michel; Togashi, Tadashi; Tono, Kensuke; Yabashi, Makina; Cammarata, Marco; Foucar, Lutz; Bourgeois, Dominique; Sliwa, Michel; Colletier, Jacques-Philippe; Schlichting, Ilme; Weik, Martin

doi:10.1038/s41467-020-14537-0

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

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Nass Kovács, Gabriela
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;
Coherent diffractive imaging, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons92083

Barends, Thomas
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;
Coherent diffractive imaging, Max Planck Institute for Medical Research, Max Planck Society;
Heme and Flavin Enzymes, Max Planck Institute for Medical Research, Max Planck Society;
Molecular chaperones, Max Planck Institute for Medical Research, Max Planck Society;
Photoreceptors, Max Planck Institute for Medical Research, Max Planck Society;
Structural Biology of Elemental Cycles, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons117878

Doak, Bruce
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;
Coherent diffractive imaging, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons117928

Nass, Karol
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;
Coherent diffractive imaging, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons183399

Roome, Christopher M.
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons95345

Shoeman, Robert L.
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;
Coherent diffractive imaging, Max Planck Institute for Medical Research, Max Planck Society;
Analytical Protein Biochemistry, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons92933

Foucar, Lutz
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons95189

Schlichting, Ilme
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;
Coherent diffractive imaging, Max Planck Institute for Medical Research, Max Planck Society;
Heme and Flavin Enzymes, Max Planck Institute for Medical Research, Max Planck Society;
Photoreceptors, Max Planck Institute for Medical Research, Max Planck Society;
Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society;
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Woodhouse, J., Nass Kovács, G., Coquelle, N., Uriarte, L. M., Adam, V., Barends, T., et al. (2020). Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy. Nature Communications, 11(741): 741, pp. 1-11. doi:10.1038/s41467-020-14537-0.

Cite as: https://hdl.handle.net/21.11116/0000-0005-A4F9-9

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.