15 オワンクラゲの緑色蛍光タンパク質クロモフォアの構造決定(口頭発表の部)
詳細
The jellyfish,Aequorea victoria, possesses a green fluorescent protein (GFP, 27kDa), which serves as the ultimate light emitter in the bioluminescence. GFP is made up of 238 amino acid residues in a single polypeptide chain and has a fluorescent chromophore emitting a green light (λ<max> = 508nm) when irradiated with an ultraviolet light. It has been supposed that the chromophore consists of an imidazolone ring, formed by a post-translational modification of the tripeptide Ser^<65>-Tyr^<66>-Gly^<67> in the primary structure. We report here the results of a spectroscopic study on a peptide fragment of GFP bearing the chromophore and on model compounds 2-4 for structural elucidation of the GFP chromophore. The mass spectroscopic analysis of the chromophore-containing peptide isolated from the protease digest of GFP establishes the chromophore formation by the dehydration-dehydrogenation mechanism in the tripeptide Ser^<65>-Tyr^<66>-Gly^<67>. The clear overlapping of the UV-vis absorption spectra of model compound 3 with those of the lysyl endopeptidase fragment of GFP indicates that the structure of the GFP chromophore consists of a 4-(4-hydroxyphenylmethylidene)imidazol-5-one ring and that the GFP chromophore has a phenolate anion structure, when GFP is in the electronically excited state. GFP lost its fluorescence completely on protease digestion. Model compound 3 also is nonfluorescent in fluid media. On the other hand, both the lysyl endopeptidase fragment and 3 became highly fluorescent in ethanol glass at 77K. These results of the fluorescent behavior of the lysyl endopeptidase fragment and 3 are explained by a competition between the photoisomerization of the exo-methylene double bond and fluorescence emission in the singlet excited state of the imidazolone chromophore. Therefore the characteristic greenish fluorescence of GFP at room temperature may be considered as being due to a restriction of the molecular motion of the chromophore within the peptide environment.
