Title |
Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP
|
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Published in |
The Journal of Physical Chemistry Letters, February 2016
|
DOI | 10.1021/acs.jpclett.5b02789 |
Pubmed ID | |
Authors |
Jacques-Philippe Colletier, Michel Sliwa, François-Xavier Gallat, Michihiro Sugahara, Virginia Guillon, Giorgio Schirò, Nicolas Coquelle, Joyce Woodhouse, Laure Roux, Guillaume Gotthard, Antoine Royant, Lucas Martinez Uriarte, Cyril Ruckebusch, Yasumasa Joti, Martin Byrdin, Eiichi Mizohata, Eriko Nango, Tomoyuki Tanaka, Kensuke Tono, Makina Yabashi, Virgile Adam, Marco Cammarata, Ilme Schlichting, Dominique Bourgeois, Martin Weik |
Abstract |
Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultra-fast 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 non-fluorescent (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 in-termediates 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 crys-tallography 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 spectro-scopic 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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