Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 3. Temperature Dependence of Proton Transfer†

Abstract

and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (ìmax = 465 nm) and green (ìmax = 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with