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Article Dans Une Revue Journal of Physical Chemistry B Année : 2015

Real-Time Monitoring of Chromophore Isomerization and Deprotonation During the Photoactivation of the Fluorescent Protein Dronpa

Résumé

Dronpa is a GFP-related photochromic fluorescent protein used as probe in superresolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic timescales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant timescales from ~100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multi-photon processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of ~0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans→cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond timescale, concomitantly to the excited-state decay. We found the deprotonation step to follow in ~10 μs and lead directly from the neutral cis intermediate to the final state.

Dates et versions

hal-01116607 , version 1 (13-02-2015)

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Citer

Dheerendra Yadav, Fabien Lacombat, Nadia Dozova, Fabrice Rappaport, Pascal Plaza, et al.. Real-Time Monitoring of Chromophore Isomerization and Deprotonation During the Photoactivation of the Fluorescent Protein Dronpa. Journal of Physical Chemistry B, 2015, Photoinduced Proton Transfer in Chemistry and Biology, 119 (6), pp.2404. ⟨10.1021/jp507094f⟩. ⟨hal-01116607⟩
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