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Journal Article
Research Support, Non-U.S. Gov't
Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the Dronpa fluorescent protein.
Journal of Physical Chemistry. B 2015 Februrary 13
The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis-trans and trans-cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright "on" and dark "off" states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete.
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