Photophysics of EGFP (E222H) Mutant, with Comparisons to Model Chromophores: Excited State pK’s, Progressions, Quenching and Exciton Interaction

A novel version of the well-known and commercially successful Green Fluorescent Protein (GFP) variant known as EGFP, with an introduced E222H mutation, was produced in this laboratory. Given the current state of hypotheses about the role of glutamate 222, and the observed dominance of the phenolate absorption with an E222H variant observed from earlier study, the new mutant was considered a natural choice to investigate more fully the acid-base behavior of the chromophore in absorption and fluorescence. The bulk of this investigation concerns fitting the excitation, emission and absorption spectra to vibrational progressions of a novel ‘q-deformed’ type at various values of pH, and protein concentration. From these data, and from temperature-dependent fluorescence lifetime data and other experiments (with lanthanide doped gels into which H/EGFP is embedded), we construct a picture of excited inter- state conversion mechanisms, and quenching mechanisms, that attempts to explain many features of the GFP system. [Figure not available: see fulltext.]