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 Fou rier transform infrared (FTIR) difference spect ra of green fluorescent and photoactive yellow proteins (GFP, PYP) employing ab initio calculations of the ground electronic state structures and vibrational spectra of their ch romophores in selected protonation states. Particular attention is addresse d to inferring the protonation states of wild-type GFP chromophore and to r econciling the measured FTIR difference spectrum with a proposed proton shu ttle mechanism in which protonated and deprotonated forms of the chromophor e are paired with corresponding charge states of a Glu222 residue shuttle t erminus. The calculated GFP IR difference spectrum obtained from the neutra l-anionic pair of chromophores is found to be in general accord with the FT IR measurements on wild-type GFP in its protonated and deprotonated farms, whereas the spectrum obtained from the zwitterionic-cationic pair of chromo phores provides a less satisfactory simulation of the data. The apparent ab sence of a carbonyl band in the measured GFP FTIR difference spectrum, a fe ature expected upon protonation of the carboxylic Glu222 residue, is reconc iled by the presence of a carbonyl mode in the imidazole ring of the neutra l chromophore which partially obscures the anticipated R-COOH Glu222 featur e in the calculated spectrum. By contrast, the corresponding carbonyl mode in the PYP chromophore is predicted to be significantly weaker and at lower frequency than in GFP, accounting in part for the presence of an unobscure d prominent R-COOH Glu46. residue carbonyl mode in the measured PYP FTIR di fference spectrum. Accordingly, the present ab initio theoretical study sup ports the predominance of neutral and anionic forms of wild-type GFP chromo phore, and it argueably reconciles the available FTIR data with a probable Glu222 terminus of the proposed proton shuttle mechanism in the protein. Ad ditional experimental studies of IR and Raman difference spectra in GFP and PYP, including particularly isotopic substitutions, are suggested to compl ement additional theoretical studies in progress.