The origin of the low steady-state fluorescence quantum yield of some blue-emitting variants of the green fluorescent protein (GFP) is investigated in single-site mutants in which the tyrosine residue at position 66 has been replaced by phenylalanine or by histidine. Time-resolved fluorescence measurements reveal excited-state lifetimes of 74 ps (Y66F) and 0.9 ns (Y66H) at room temperature that increase to values close to the radiative limit as the temperature is lowered. These short lifetimes are explained by temperature-dependent internal conversion. The pronounced difference between the room-temperature lifetimes of the two mutants suggests that hydrogen bonding of the distal aromatic ring plays a more important role than tight packing in the fixation of the chromophore.
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