TY - JOUR
T1 - Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP
AU - Colletier, Jacques Philippe
AU - Sliwa, Michel
AU - Gallat, François Xavier
AU - Sugahara, Michihiro
AU - Guillon, Virginia
AU - Schirò, Giorgio
AU - Coquelle, Nicolas
AU - Woodhouse, Joyce
AU - Roux, Laure
AU - Gotthard, Guillaume
AU - Royant, Antoine
AU - Uriarte, Lucas Martinez
AU - Ruckebusch, Cyril
AU - Joti, Yasumasa
AU - Byrdin, Martin
AU - Mizohata, Eiichi
AU - Nango, Eriko
AU - Tanaka, Tomoyuki
AU - Tono, Kensuke
AU - Yabashi, Makina
AU - Adam, Virgile
AU - Cammarata, Marco
AU - Schlichting, Ilme
AU - Bourgeois, Dominique
AU - Weik, Martin
N1 - Funding Information:
The authors are grateful to Leonard Chavas and So Iwata for technical support at SACLA. J.P.C. thanks Duilio Cascio, Michael Sawaya, and David Eisenberg for stimulating discussions and continuing support. M.Sl. is grateful to Jean-Pierre Verwaerde and Julien Dubois for technical support in the rotating cell design and time-resolved experiments. The XFEL experiments were carried out at BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; Proposal No. 2013B8046), and the synchrotron experiments at ID29 of ESRF, under long-term projects MX1464, MX1583, and MX1676 (IBS BAG). We warmly thank the SACLA and ESRF staff for assistance. The study was supported by a grant from the CNRS (PEPS SASLELX) to M.W., and ANR grants to M.W., M.C., M.Sl. (BioXFEL) and to D.B. (NoBleach). The Cryobench is a platform of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from ESRF, FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01). 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 to M.Sl.
Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/3/3
Y1 - 2016/3/3
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.jpclett.5b02789
DO - 10.1021/acs.jpclett.5b02789
M3 - Article
C2 - 26866390
AN - SCOPUS:84960145363
SN - 1948-7185
VL - 7
SP - 882
EP - 887
JO - Journal of Physical Chemistry Letters
JF - Journal of Physical Chemistry Letters
IS - 5
ER -