The structure of the chromophore within DsRed, a red fluorescent protein from coral

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Abstract
DsRed, a-brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea, A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed, After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming -C=N-C=O at the 2-position of the imidazolidinone, This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C=N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C=N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.