Raman study of chromophore states in photochromic fluorescent proteins

The photophysical mechanism underlying the photochromic behavior of green fluorescent protein (GFP) mutants is investigated by means of preresonant Raman spectroscopy and model calculations. The studied molecules are reversibly switchable fluorophores that can be repeatedly converted between fluorescent and nonfluorescent states by irradiation and are attracting a broad interest for a number of new applications. Experimental results on chemically synthesized isolated chromophores are analyzed within a density functional theory approach that allows us to link the detected vibrational modes to specific ground-state configurations before and after photoconversion. These data are compared to results obtained for the case of complete folded proteins including the same chromophores. Our results highlight the impact of chromophore cis-trans isomerization and protonation state in the photophysics of these proteins and provide useful guidelines for novel mutant design.