CHROMOPHORE-CONTAINING PEPTIDES FROM AEQUOREA AND RENILLA GREEN-FLUORESCENT PROTEINS

Bioluminescence in many coelenterates involves an energy transfer reaction wherein a green-fluorescent protein (GFP) functions as the in vivo light emitter. Native GFPs exhibit species-specific absorption, fluorescence excitation, and fluorescence emission spectra. The hydrozoan Aequorea aequorea and the anthozoan Renilla reniformis have GFPs with quite different native absorption spectra; however, the denatured proteins are spectrally identical. Both denatured GFPs exhibit identical pH-dependent absorption spectra. Furthermore, a small papain-derived chromophore-containing peptide, that retains the absorption characteristics of denatured GFP, can be isolated from either species. The above spectral evidence implies that the two GFPs contain identical chromophores.;An improved method for purifying the Aequorea GFP papain limit-digest chromopeptide by a combination of chromatography on Bio-Gel P-2 and HPLC was developed. The high sensitivity of this methodology made possible the detection and separation not only of the limit-digest chromopeptide but also of previously undetected minor chromopeptides. At least one of these minor peptides was slightly larger than the limit-digest chromopeptide; other peptides showed evidence of chromophore degradation.;The Aequorea GFP chromophore is incorporated within a covalently modified pentapeptide, the sequence of which is phenylalanine-glycine-dehydrotyrosine-valine-glutamate. The sequence of this limit-digest chromopeptide was determined by treatment with several other proteases including carboxypeptidase A, which hydrolyzed phenylalanine and valine from the peptide, and pronase, which hydrolyzed glutamate and phenylalanine.;A papain limit-digest chromopeptide was also purified from a Renilla GFP papain digestion. The Renilla GFP chromopeptide differs from the Aequorea GFP chromopeptide in that it contains no glutamate or valine; instead it has two equivalents of aspartate and one equivalent of arginine.;A chromophore-containing peptide released from Aequorea GFP was initially isolated by O. Shimomura (1979, FEBS Letters, 104(2):220-222). He determined that the chromophore of Aequorea GFP was 4-p-hydroxybenzylidene-5-imidazolone.