Variants of Green Fluorescent Protein (GFP) are in widespread use as genetically encoded labels for biological imaging. Excitation of its neutral phenolic ground-state chromophore populates an emissive anionic phenolate state via excited-state proton transfer (ESPT) to E222 along a molecular wire. Computational studies indicate that ESPT fits an electronically adiabatic rate expression. Although reactions that operate close to the adiabatic limit can be sensitive to environmental dipolar relaxation, little is known about the role of such relaxation during ESPT in GFP. We present the first experimental evidence that dipolar relaxation of the protein matrix occurs during ESPT in response to the change in charge density distribution in the excited-state GFP chromophore and is a key determinant of the reaction pathway. Using fluorescence spectroscopy, we excited along the red edge of the neutral phenolic ground-state absorption band of several GFP variants with differing chromophore environments. Instead of resulting in a significant red shift of the center of spectral mass (CSM) of the emission spectra common for biological chromophores such as tryptophan, the CSM of each GFP variant remains almost unchanged as a function of excitation wavelength. This is consistent with each subpopulation that is selectively excited along the red edge reaching the same fully relaxed state before emission of a photon. The kinetics of environmental dipolar relaxation are therefore on the same subnanosecond time scale as ESPT, which provides an explanation for its adiabatic nature and could inform the rational design of novel fluorescent proteins with tailored photophysics.
Kurttila et al. (Sun,) studied this question.