title: Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.
abstract: Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Jacques-Philippe
authors: JP
authors: Colletier
authors: Université de Lille , CNRS, UMR 8516, LASIR, Laboratoire de Spectrochimie Infrarouge et Raman, F59 000 Lille, France.
authors: Michel
authors: M
authors: Sliwa
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: François-Xavier
authors: FX
authors: Gallat
authors: RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors: Michihiro
authors: M
authors: Sugahara
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Virginia
authors: V
authors: Guillon
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Giorgio
authors: G
authors: Schirò
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Nicolas
authors: N
authors: Coquelle
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Joyce
authors: J
authors: Woodhouse
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Laure
authors: L
authors: Roux
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.|The European Synchrotron Radiation Facility (ESRF) , 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France.
authors: Guillaume
authors: G
authors: Gotthard
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.|The European Synchrotron Radiation Facility (ESRF) , 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France.
authors: Antoine
authors: A
authors: Royant
authors: Université de Lille , CNRS, UMR 8516, LASIR, Laboratoire de Spectrochimie Infrarouge et Raman, F59 000 Lille, France.
authors: Lucas Martinez
authors: LM
authors: Uriarte
authors: Université de Lille , CNRS, UMR 8516, LASIR, Laboratoire de Spectrochimie Infrarouge et Raman, F59 000 Lille, France.
authors: Cyril
authors: C
authors: Ruckebusch
authors: Japan Synchrotron Radiation Research Institute , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan.
authors: Yasumasa
authors: Y
authors: Joti
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Martin
authors: M
authors: Byrdin
authors: Department of Applied Chemistry, Graduate School of Engineering, Osaka University , Osaka 565-0871, Japan.
authors: Eiichi
authors: E
authors: Mizohata
authors: RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors: Eriko
authors: E
authors: Nango
authors: RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors: Tomoyuki
authors: T
authors: Tanaka
authors: Japan Synchrotron Radiation Research Institute , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan.
authors: Kensuke
authors: K
authors: Tono
authors: RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors: Makina
authors: M
authors: Yabashi
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Virgile
authors: V
authors: Adam
authors: Department of Physics, UMR UR1-CNRS 6251, University of Rennes 1 , Rennes, France.
authors: Marco
authors: M
authors: Cammarata
authors: Max-Planck-Institut für medizinische Forschung , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors: Ilme
authors: I
authors: Schlichting
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Dominique
authors: D
authors: Bourgeois
authors: Institut de Biologie Structurale , Université de Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France.
authors: Martin
authors: M
authors: Weik
mesh_terms: D003461:Crystallography / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D008164:Luminescent Proteins