10.1021/jp507094f.s001
Dheerendra Yadav
Dheerendra
Yadav
Fabien Lacombat
Fabien
Lacombat
Nadia Dozova
Nadia
Dozova
Fabrice Rappaport
Fabrice
Rappaport
Pascal Plaza
Pascal
Plaza
Agathe Espagne
Agathe
Espagne
Real-Time
Monitoring of Chromophore Isomerization
and Deprotonation during the Photoactivation of the Fluorescent Protein
Dronpa
American Chemical Society
2015
photochromic
chromophore
anisotropy
picosecond time scale
deprotonation
UV
absorption spectroscopy study
ci
photoactivation
Fluorescent Protein DronpaDronpa
time scales
GFP
isomerization
2015-02-12 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Real_Time_Monitoring_of_Chromophore_Isomerization_and_Deprotonation_during_the_Photoactivation_of_the_Fluorescent_Protein_Dronpa/2196655
Dronpa
is a photochromic green fluorescent protein (GFP) homologue
used as a probe in super-resolution 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 time scales 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 time scales 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
multiphoton 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 time scale,
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.