The mechanism of green fluorescent protein (GFP) chromophore formation is still not clearly defined. Two mechanisms have been proposed: cyclisation-dehydration-oxidation (Mechanism A) and cyclisation-oxidation-dehydration (Mechanism B). To distinguish between these mechanisms, we generated a non-fluorescent mutant of GFP, S65T/G67A-GFP. This mutant folds to a stable, native-like structure but lacks fluorescence due to interruption of the chromophore maturation process. Mass spectrometric analysis of peptides derived from this mutant reveal that chromophore formation follows only mechanism A, but that the final oxidation reaction is suppressed. This result is unexpected within the pool of examined GFP mutants, since for the wild-type GFP, there is strong support for mechanism B.
Keywords: S65T mutant; S65T/G67A mutant; chromophore maturation; green fluorescent protein; ms/ms spectrometry.
© 2018 Federation of European Biochemical Societies.