skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

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 in this paper 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.

Authors:
 [1]; ORCiD logo [2];  [1];  [1]; ORCiD logo [1];  [3];  [4];  [3];  [1];  [3];  [1];  [4];  [1];  [1];  [4];  [1];  [4];  [3]; ORCiD logo [5];  [3] more »;  [4];  [3];  [6];  [3];  [4];  [6];  [3];  [4];  [2];  [3];  [1]; ORCiD logo [7];  [6]; ORCiD logo [1];  [4];  [1]; ORCiD logo [1];  [4]; ORCiD logo [1] « less
  1. Univ. of Grenoble (France). Inst. de Biologie Structurale
  2. Univ. de Lille (France). Lab. de Spectrochimie Infrarouge et Raman
  3. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
  4. Max-Planck-Inst. fur medizinische Forschung, Heidelberg (Germany)
  5. Max-Planck-Inst. fur biophysikalische Chemie, Gottingen (Germany)
  6. Univ. Paris-Sud, Orsay (France); Univ. Paris-Saclay, Orsay (France). Lab. de Chimie-Physique
  7. Univ. of Rennes, Rennes (France). Dept. of Physics
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); European Union (EU)
OSTI Identifier:
1426209
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
Nature Chemistry
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 1755-4330
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
77 NANOSCIENCE AND NANOTECHNOLOGY; 59 BASIC BIOLOGICAL SCIENCES; Nanocrystallography; Photobiology; Protein structure predictions

Citation Formats

Coquelle, Nicolas, Sliwa, Michel, Woodhouse, Joyce, Schirò, Giorgio, Adam, Virgile, Aquila, Andrew, Barends, Thomas R. M., Boutet, Sébastien, Byrdin, Martin, Carbajo, Sergio, De la Mora, Eugenio, Doak, R. Bruce, Feliks, Mikolaj, Fieschi, Franck, Foucar, Lutz, Guillon, Virginia, Hilpert, Mario, Hunter, Mark S., Jakobs, Stefan, Koglin, Jason E., Kovacsova, Gabriela, Lane, Thomas J., Lévy, Bernard, Liang, Mengning, Nass, Karol, Ridard, Jacqueline, Robinson, Joseph S., Roome, Christopher M., Ruckebusch, Cyril, Seaberg, Matthew, Thepaut, Michel, Cammarata, Marco, Demachy, Isabelle, Field, Martin, Shoeman, Robert L., Bourgeois, Dominique, Colletier, Jacques-Philippe, Schlichting, Ilme, and Weik, Martin. Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography. United States: N. p., 2017. Web. doi:10.1038/nchem.2853.
Coquelle, Nicolas, Sliwa, Michel, Woodhouse, Joyce, Schirò, Giorgio, Adam, Virgile, Aquila, Andrew, Barends, Thomas R. M., Boutet, Sébastien, Byrdin, Martin, Carbajo, Sergio, De la Mora, Eugenio, Doak, R. Bruce, Feliks, Mikolaj, Fieschi, Franck, Foucar, Lutz, Guillon, Virginia, Hilpert, Mario, Hunter, Mark S., Jakobs, Stefan, Koglin, Jason E., Kovacsova, Gabriela, Lane, Thomas J., Lévy, Bernard, Liang, Mengning, Nass, Karol, Ridard, Jacqueline, Robinson, Joseph S., Roome, Christopher M., Ruckebusch, Cyril, Seaberg, Matthew, Thepaut, Michel, Cammarata, Marco, Demachy, Isabelle, Field, Martin, Shoeman, Robert L., Bourgeois, Dominique, Colletier, Jacques-Philippe, Schlichting, Ilme, & Weik, Martin. Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography. United States. doi:10.1038/nchem.2853.
Coquelle, Nicolas, Sliwa, Michel, Woodhouse, Joyce, Schirò, Giorgio, Adam, Virgile, Aquila, Andrew, Barends, Thomas R. M., Boutet, Sébastien, Byrdin, Martin, Carbajo, Sergio, De la Mora, Eugenio, Doak, R. Bruce, Feliks, Mikolaj, Fieschi, Franck, Foucar, Lutz, Guillon, Virginia, Hilpert, Mario, Hunter, Mark S., Jakobs, Stefan, Koglin, Jason E., Kovacsova, Gabriela, Lane, Thomas J., Lévy, Bernard, Liang, Mengning, Nass, Karol, Ridard, Jacqueline, Robinson, Joseph S., Roome, Christopher M., Ruckebusch, Cyril, Seaberg, Matthew, Thepaut, Michel, Cammarata, Marco, Demachy, Isabelle, Field, Martin, Shoeman, Robert L., Bourgeois, Dominique, Colletier, Jacques-Philippe, Schlichting, Ilme, and Weik, Martin. Mon . "Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography". United States. doi:10.1038/nchem.2853. https://www.osti.gov/servlets/purl/1426209.
@article{osti_1426209,
title = {Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography},
author = {Coquelle, Nicolas and Sliwa, Michel and Woodhouse, Joyce and Schirò, Giorgio and Adam, Virgile and Aquila, Andrew and Barends, Thomas R. M. and Boutet, Sébastien and Byrdin, Martin and Carbajo, Sergio and De la Mora, Eugenio and Doak, R. Bruce and Feliks, Mikolaj and Fieschi, Franck and Foucar, Lutz and Guillon, Virginia and Hilpert, Mario and Hunter, Mark S. and Jakobs, Stefan and Koglin, Jason E. and Kovacsova, Gabriela and Lane, Thomas J. and Lévy, Bernard and Liang, Mengning and Nass, Karol and Ridard, Jacqueline and Robinson, Joseph S. and Roome, Christopher M. and Ruckebusch, Cyril and Seaberg, Matthew and Thepaut, Michel and Cammarata, Marco and Demachy, Isabelle and Field, Martin and Shoeman, Robert L. and Bourgeois, Dominique and Colletier, Jacques-Philippe and Schlichting, Ilme and Weik, Martin},
abstractNote = {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 in this paper 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.},
doi = {10.1038/nchem.2853},
journal = {Nature Chemistry},
number = 1,
volume = 10,
place = {United States},
year = {2017},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 18 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Fluorescent proteins for live-cell imaging with super-resolution
journal, January 2014

  • Nienhaus, Karin; Ulrich Nienhaus, G.
  • Chem. Soc. Rev., Vol. 43, Issue 4
  • DOI: 10.1039/C3CS60171D

Chromophore Protonation State Controls Photoswitching of the Fluoroprotein asFP595
journal, March 2008


Use of a `caged' analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography
journal, October 2007

  • Colletier, Jacques-Philippe; Royant, Antoine; Specht, Alexandre
  • Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 11
  • DOI: 10.1107/S0907444907044472

Photoisomerization by Hula-Twist:  A Fundamental Supramolecular Photochemical Reaction
journal, July 2001

  • Liu, Robert S. H.
  • Accounts of Chemical Research, Vol. 34, Issue 7
  • DOI: 10.1021/ar000165c

Structural basis for reversible photoswitching in Dronpa
journal, July 2007

  • Andresen, M.; Stiel, A. C.; Trowitzsch, S.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 32
  • DOI: 10.1073/pnas.0700629104

Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy
journal, August 2008

  • Andresen, Martin; Stiel, Andre C.; Fölling, Jonas
  • Nature Biotechnology, Vol. 26, Issue 9
  • DOI: 10.1038/nbt.1493

Advances in fluorescence labeling strategies for dynamic cellular imaging
journal, June 2014


Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif
journal, January 2016

  • Pandelieva, Antonia T.; Baran, Miranda J.; Calderini, Guido F.
  • ACS Chemical Biology, Vol. 11, Issue 2
  • DOI: 10.1021/acschembio.5b00774

Crystallographic data processing for free-electron laser sources
journal, June 2013

  • White, Thomas A.; Barty, Anton; Stellato, Francesco
  • Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 7
  • DOI: 10.1107/S0907444913013620

The Green Fluorescent Protein
journal, June 1998


Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations
journal, November 2008

  • Adam, V.; Lelimousin, M.; Boehme, S.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 47
  • DOI: 10.1073/pnas.0805949105

Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%
journal, January 2012

  • Goedhart, Joachim; von Stetten, David; Noirclerc-Savoye, Marjolaine
  • Nature Communications, Vol. 3, Issue 1
  • DOI: 10.1038/ncomms1738

Structure and rotation barriers for ground and excited states of the isolated chromophore of the green fluorescent protein
journal, November 1998

  • Voityuk, Alexander A.; Michel-Beyerle, Maria-Elisabeth; Rösch, Notker
  • Chemical Physics Letters, Vol. 296, Issue 3-4
  • DOI: 10.1016/S0009-2614(98)01035-5

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa
journal, February 2013

  • Warren, Mark M.; Kaucikas, Marius; Fitzpatrick, Ann
  • Nature Communications, Vol. 4, Issue 1
  • DOI: 10.1038/ncomms2460

Protonic Gating of Excited-State Twisting and Charge Localization in GFP Chromophores: A Mechanistic Hypothesis for Reversible Photoswitching
journal, February 2010

  • Olsen, Seth; Lamothe, Kristina; Martínez, Todd J.
  • Journal of the American Chemical Society, Vol. 132, Issue 4
  • DOI: 10.1021/ja907447k

CASS—CFEL-ASG software suite
journal, October 2012

  • Foucar, Lutz; Barty, Anton; Coppola, Nicola
  • Computer Physics Communications, Vol. 183, Issue 10
  • DOI: 10.1016/j.cpc.2012.04.023

Low-Temperature Chromophore Isomerization Reveals the Photoswitching Mechanism of the Fluorescent Protein Padron
journal, October 2011

  • Regis Faro, Aline; Carpentier, Philippe; Jonasson, Gabriella
  • Journal of the American Chemical Society, Vol. 133, Issue 41
  • DOI: 10.1021/ja207001y

rsEGFP2 enables fast RESOLFT nanoscopy of living cells
journal, December 2012


Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams
journal, April 2015

  • Coquelle, Nicolas; Brewster, Aaron S.; Kapp, Ulrike
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 5
  • DOI: 10.1107/S1399004715004514

Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy
journal, November 2009

  • Fang, Chong; Frontiera, Renee R.; Tran, Rosalie
  • Nature, Vol. 462, Issue 7270
  • DOI: 10.1038/nature08527

Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein
journal, May 2016


Phototransformable fluorescent proteins: Future challenges
journal, June 2014


Excited State Dynamics of the Green Fluorescent Protein on the Nanosecond Time Scale
journal, May 2011

  • Jonasson, Gabriella; Teuler, Jean-Marie; Vallverdu, Germain
  • Journal of Chemical Theory and Computation, Vol. 7, Issue 6
  • DOI: 10.1021/ct200150r

The Role of the Protein Matrix in Green Fluorescent Protein Fluorescence
journal, January 2006


Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm
journal, January 2016

  • El Khatib, Mariam; Martins, Alexandre; Bourgeois, Dominique
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep18459

Structure and mechanism of the reversible photoswitch of a fluorescent protein
journal, August 2005

  • Andresen, M.; Wahl, M. C.; Stiel, A. C.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 37
  • DOI: 10.1073/pnas.0502772102

Real-Time Monitoring of Chromophore Isomerization and Deprotonation during the Photoactivation of the Fluorescent Protein Dronpa
journal, August 2014

  • Yadav, Dheerendra; Lacombat, Fabien; Dozova, Nadia
  • The Journal of Physical Chemistry B, Vol. 119, Issue 6
  • DOI: 10.1021/jp507094f

The Coherent X-ray Imaging instrument at the Linac Coherent Light Source
journal, April 2015

  • Liang, Mengning; Williams, Garth J.; Messerschmidt, Marc
  • Journal of Synchrotron Radiation, Vol. 22, Issue 3
  • DOI: 10.1107/S160057751500449X

Photocycle populations with femtosecond excitation of crystalline photoactive yellow protein
journal, June 2016


Shedding light on the dark and weakly fluorescent states of green fluorescent proteins
journal, May 1999

  • Weber, W.; Helms, V.; McCammon, J. A.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 11
  • DOI: 10.1073/pnas.96.11.6177

Excited state reactions in fluorescent proteins
journal, January 2009

  • Meech, Stephen R.
  • Chemical Society Reviews, Vol. 38, Issue 10
  • DOI: 10.1039/b820168b

CFEL–ASG Software Suite ( CASS ): usage for free-electron laser experiments with biological focus
journal, June 2016


Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP
journal, February 2016

  • Colletier, Jacques-Philippe; Sliwa, Michel; Gallat, François-Xavier
  • The Journal of Physical Chemistry Letters, Vol. 7, Issue 5
  • DOI: 10.1021/acs.jpclett.5b02789

Improved Estimation of Structure-Factor Difference Amplitudesfrom Poorly Accurate Data
journal, September 1997

  • Ursby, T.; Bourgeois, D.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 53, Issue 5
  • DOI: 10.1107/S0108767397004522

Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments
journal, June 2017

  • Schirò, Giorgio; Woodhouse, Joyce; Weik, Martin
  • Journal of Applied Crystallography, Vol. 50, Issue 3
  • DOI: 10.1107/S1600576717006264

Difference refinement: obtaining differences between two related structures
journal, September 1995

  • Terwilliger, T. C.; Berendzen, J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 51, Issue 5
  • DOI: 10.1107/S0907444994013247

Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation
journal, September 2015


    Works referencing / citing this record:

    Megahertz data collection from protein microcrystals at an X-ray free-electron laser
    journal, August 2018

    • Grünbein, Marie Luise; Bielecki, Johan; Gorel, Alexander
    • Nature Communications, Vol. 9, Issue 1
    • DOI: 10.1038/s41467-018-05953-4

    Room-temperature photo-induced martensitic transformation in a protein crystal
    journal, May 2019


    Specific radiation damage is a lesser concern at room temperature
    journal, June 2019


    Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin
    journal, July 2019

    • Nass Kovacs, Gabriela; Colletier, Jacques-Philippe; Grünbein, Marie Luise
    • Nature Communications, Vol. 10, Issue 1
    • DOI: 10.1038/s41467-019-10758-0

    Comparative study of two near-infrared coumarin–BODIPY dyes for bioimaging and photothermal therapy of cancer
    journal, January 2019

    • Zhang, Yuandong; Song, Nan; Li, Yuanyuan
    • Journal of Materials Chemistry B, Vol. 7, Issue 30
    • DOI: 10.1039/c9tb01165j