Green fluorescent protein ground states: The influence of a second protonation site near the chromophore

The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.