Thefast-switching M159T mutant of the reversibly photoswitchablefluorescent protein Dronpa has an enhanced yield for the on-to-offreaction. The forward and reverse photoreactions proceed via cis–transand trans–cis photoisomerization, yet protonation and deprotonationof the hydroxyphenyl oxygen of the chromophore is responsible forthe 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, staticFTIR difference measurements of the Dronpa-M159T mutant correspondvery closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinonechromophore 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 radiativedecay with characteristic time constants of 1.9 ps, 185 ps, and 1.1ns, and additionally reveals spectral changes belonging to the speciesdecaying with a 1.1 ns time constant, associated with both proteinand chromophore modes. A 1 ms measurement of the on state identifiesbleach features that correspond to those seen in the static off-minus-onFourier transform infrared (FTIR) difference spectrum, indicatingthat thermal protonation of the hydroxyphenyl oxygen proceeds withinthis time window. Blue excitation of the off state directly resolvesthe formation of the primary photoproduct with 0.6 and 14 ps timeconstants, which is stable on the nanosecond time scale. Assignmentof the primary photoproduct to the cis neutral chromophore in theelectronic ground state is supported by the frequency positions expectedrelative to those for the nonplanar distorted geometry for the offstate. A 1 ms measurement of the off state corresponds closely withthe on-minus-off FTIR difference spectrum, indicating thermal deprotonationand rearrangement of the Arg66 side chain to be complete. [ABSTRACT FROM AUTHOR]