Ultrafast proton transfer in the green fluorescent protein: Analysing the instantaneous emission at product state wavelengths

Knowledge of the photophysics of the green fluorescent protein (GFP) underpins its exploitation in bioimaging. Some aspects of the fluorescence dynamics remain incompletely understood. The wavelength resolved ultrafast fluorescence of wtGFP and the mutant T203V are studied here as a function of excitation wavelength. These results allow us to address some of these unexplained phenomena. The apparently instantaneous component observed in the emission of the anionic state, which is formed from the neutral state, is shown to arise from a contribution of the underlying and unusually broad neutral state emission. The entire emission spectrum can be fit by a sum of only two spectra, suggesting that no additional emissive intermediates contribute. Thus the non-exponential kinetics are likely to arise from an inhomogeneous distribution of ground state structures.