Chromophore Protonation States and the Proton Shuttle Mechanism in Green Fluorescent Protein: Inferences Drawn from ab Initio Theoretical Studies of Chemical Structures and Vibrational Spectra

Assignments are provided of prominent features in the recently measured Fourier transform infrared (FTIR) difference spectra of green fluorescent and photoactive yellow proteins (GFP, PYP) employing ab initio calculations of the ground electronic state structures and vibrational spectra of their chromophores in selected protonation states. Particular attention is addressed to inferring the protonation states of wild-type GFP chromophore and to reconciling the measured FTIR difference spectrum with a proposed proton shuttle mechanism in which protonated and deprotonated forms of the chromophore are paired with corresponding charge states of a Glu222 residue shuttle terminus. The calculated GFP IR difference spectrum obtained from the neutral-anionic pair of chromophores is found to be in general accord with the FTIR measurements on wild-type GFP in its protonated and deprotonated forms, whereas the spectrum obtained from the zwitterionic-cationic pair of chromophores provides a less satisfactory simulation...