The jellyfish Aequorea victoria possesses in the margin of its umbrella a green f luorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish f luorescence ( max ‫؍‬ 508 nm) when irradiated with long ultraviolet light. The f luorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser 65 -Tyr 66 -Gly 67 -. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which f luorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

Time-resolved fluorescence measurements at 275 K show that the excited-state lifetime of a model chromophore of the green fluorescent protein (GFP) substantially increases from subpicoseconds in low-viscosity solvents such as ethanol (η = 1.7 cP) to 30 ps in glycerol (η = 9.9 × 103 cP) and reaches 2.1 ns in glycerol glass at 150 K. At high temperatures the similarity of excited-state decay and ground-state recovery kinetics indicates internal conversion being responsible for the short fluorescence lifetimes. Their viscosity dependence reflects on a motion with a considerable amplitude that is damped by viscous drag and outweighs thermal activation as is concluded from measurements at different temperatures. In solution the neutral and the anionic forms of the model chromophore are similarly nonfluorescent in contrast to wild-type GFP and mutants where the deprotonated form is hardly undergoing internal conversion. Thus, the protein selectively restricts motional degrees of freedom of the chromophore in specific protonation states.