Steady-state and time-resolved spectroscopic studies of green-to-red photoconversion of fluorescent protein Dendra2

The excited state dynamics and green-to-red photoconversion (PC) of the monomer fluorescent protein Dendra2 were studied using time resolved pump-probe and double-pulsed excitation experiments. A linear dependence of the PC efficiency on the pump power is demonstrated when exciting the neutral green form at 400 nm as well as anionic green form at either 458 or 488 nm. Rather large PC quantum yields of ∼1.5 × 10−3 for the neutral form and ∼6 × 10−5 for the anionic form are determined from exposure-dependent PC saturation experiments. At the same time, no dependence of the PC rate on the time delay between the pulses is observed, as well as no significant difference between PC via pulsed or continuous-wave (CW) excitation. Our wavelength- and pH-dependent studies of the PC process suggest that the Dendra2 green-to-red conversion occurs following the absorption of one photon, directly from the excited state of each (neutral and anionic) form of the green protein without intermediate excited states or species requiring absorption of additional photons.