Observation of excited-state proton transfer in green fluorescent protein using ultrafast vibrational spectroscopy

The photodynamics of wtGFP have been studied by ultrafast time-resolved infrared spectroscopy (TIR). In addition to the expected bleaching and transient infrared absorption of bands associated with the chromophore, we observe the dynamics of the proton relay reaction in the protein. Protonation of a protein carboxylate group occurs on the tens of picoseconds time scale following photoexcitation. Comparison with data for mutant GFPs, in which excited-state proton transfer has been disabled, supports the assignment of the carboxylate to the side chain of E222, a component of the hydrogen bonding network that links the two ends of the chromophore. The TIR data show that the rate-limiting step in the proton relay is deprotonation of the chromophore.