Excited state dynamics of photoswitchable fluorescent protein Padron

The key events in the light-induced switching mechanism of the photochromic green fluorescent protein Padron have been investigated by employing femtosecond fluorescence up-conversion, femtosecond transient absorption, and time-correlated single photon counting techniques. In contrast to Dronpa, excitation of protein's neutral state at 395 nm triggers an efficient and complex photoswitching to a dark state whereas irradiation with 495 nm light reverses the protein to its initial state restoring the bright fluorescence. On the basis of the kinetics observed upon irradiation of the chromophore in the protonated state, we suggest that the switching mechanism consists of a light-initiated excited state process (presumably ESPT) with a time constant of 1 ps producing an unstable intermediate state, tentatively assigned to the excited state of the cis-anionic form, that is followed by a cis- to trans- isomerization (14.5 ps) forming the trans-anionic state in which the dark chromophore resides. In the trans-state, the protonation equilibrium strongly favors the anionic form. Consequently, upon excitation of the formed anionic species a trans-cis isomerization of the chromophore was found to occur with a time constant as fast as 5.2 ps switching the chromophore quantitatively to the bright (anionic) state.