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