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Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography

Nature Chemistry volume 10, pages 31–37 (2018)Cite this article

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Abstract

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

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Figure 1: Chromophore isomers and protonation states of rsEGFP2.
Figure 2: Femtosecond TA spectroscopy of rsEGFP2 in solution.
Figure 3: Model for the off-to-on photoswitching of rsEGFP2.
Figure 4: rsEGFP2 in the electronically excited S1 state.
Figure 5: Chromophore twisting is accompanied by a downward movement of the central α-helix along the barrel axis.

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Acknowledgements

The study was supported by a grant from the CNRS (PEPS SASLELX) to M.W., and an ANR grant to M.W., M.C. and M.Sliwa (ANR-15-CE32-0004 BioXFEL). Use of LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract no. DE-AC02-76SF00515. We acknowledge support from the Max Planck Society. This work used the Multistep Protein Purification Platform (MP3) of the Grenoble Instruct Center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The Chevreul Institute (FR 2638), the Ministère de l'Enseignement Supérieur et de la Recherche, the Région Nord-Pas de Calais and FEDER are acknowledged for financial support. G.K. acknowledges support from the European Union under the program FP7-PEOPLE-2011-ITN NanoMem (project number 317079). J.W. acknowledges support from the CEA through the IRTELIS PhD program.

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Author notes

  1. Nicolas Coquelle, Michel Sliwa, Joyce Woodhouse, Giorgio Schirò and Virgile Adam: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Biologie Structurale, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, F-38044, France

    Nicolas Coquelle, Joyce Woodhouse, Giorgio Schirò, Virgile Adam, Martin Byrdin, Eugenio De la Mora, Mikolaj Feliks, Franck Fieschi, Virginia Guillon, Michel Thepaut, Martin Field, Dominique Bourgeois, Jacques-Philippe Colletier & Martin Weik

  2. Laboratoire de Spectrochimie Infrarouge et Raman, Université de Lille, CNRS, UMR 8516, LASIR, Lille, F59 000, France

    Michel Sliwa & Cyril Ruckebusch

  3. Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, 94025, California, USA

    Andrew Aquila, Sébastien Boutet, Sergio Carbajo, Mark S. Hunter, Jason E. Koglin, Thomas J. Lane, Mengning Liang, Joseph S. Robinson & Matthew Seaberg

  4. Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, Heidelberg, 69120, Germany

    Thomas R. M. Barends, R. Bruce Doak, Lutz Foucar, Mario Hilpert, Gabriela Kovacsova, Karol Nass, Christopher M. Roome, Robert L. Shoeman & Ilme Schlichting

  5. Department of NanoBiophotonics, Max-Planck-Institut für biophysikalische Chemie, 37077 Göttingen, Germany

    Stefan Jakobs

  6. Laboratoire de Chimie-Physique, CNRS/University Paris-Sud, University Paris-Saclay, UMR 8000, Orsay, 91405, France

    Bernard Lévy, Jacqueline Ridard & Isabelle Demachy

  7. Department of Physics, University of Rennes 1, UMR UR1-CNRS 6251, Rennes, France

    Marco Cammarata

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  1. Nicolas Coquelle
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Contributions

M.W., D.B., J.-P.C. and I.S. designed the research. S.J. provided the rsEGFP2 plasmid. J.W., V.A., V.G., M.T. and F.F. expressed and purified the WT protein. V.A. cloned, expressed and purified the protein variant and V.A. and D.B. analysed it. J.W., V.G. and I.S. carried out the microcrystallization. G.S. and M.B. spectroscopically characterized the microcrystals. R.L.S. and G.S. designed and optimized the pre-illumination device. M.Feliks and M.Field carried out the excited-state QM/MM simulations. J.R., M.L. and I.D. carried out the excited-state classical MD simulations. M.S. and C.R. carried out time-resolved absorption spectroscopy. M.C. and G.S. carried out the steady-state absorption spectroscopy with femtosecond excitation. N.C, M.Sliwa, J.W., V.A., A.A., T.R.M.B., S.B., S.C., E.D.l.M., R.B.D., L.F., M.Hilpert, M.S.Hunter, J.E.K., G.K., T.J.L., M.L., K.N., J.S.R., C.M.R., M.Seaberg, M.C., R.L.S., J.P.C., I.S. and M.W. carried out the SFX experiments. R.L.S, R.B.D. and G.K. performed sample injections. J.S.R. and M.C. aligned and controlled the pump laser. A.A., S.B., M.S.Hunter, J.E.K. and M.L. prepared and performed data collection. C.M.R., M.Hilpert, L.F., N.C., T.R.M.B. and J.-P.C. carried out the online SFX data processing. N.C. and J.-P.C. carried out the offline SFX data processing and structure refinement, with input from T.R.M.B., K.N., I.S., D.B. and M.W. J.-P.C., G.S., R.L.S., J.W., M.S., M.C., M.Field, J.R. and B.L. wrote the Supplementary Information with input from M.W., N.C., T.R.M.B., I.S. and D.B. M.W., D.B. and I.S. wrote the manuscript with input from T.R.M.B., M.Field, J.-P.C., M.S. and I.D.

Corresponding authors

Correspondence to Jacques-Philippe Colletier, Ilme Schlichting or Martin Weik.

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Coquelle, N., Sliwa, M., Woodhouse, J. et al. Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography. Nature Chem 10, 31–37 (2018). https://doi.org/10.1038/nchem.2853

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