Niwa H, Inouye S, Hirano T, Matsuno T, Kojima S, Kubota M, Ohashi M, Tsuji F I
Department of Applied Physics and Chemistry, University of Electro-Communications, Tokyo, Japan.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13617-22. doi: 10.1073/pnas.93.24.13617.
The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent 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 fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. 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 fluorescence 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.
维多利亚多管水母在其伞边缘拥有一种绿色荧光蛋白（GFP，27 kDa），该蛋白在动物的生物发光反应中作为最终的发光体。该蛋白由一条单多肽链中的238个氨基酸残基组成，在用长紫外光照射时会产生绿色荧光（最大波长 = 508 nm）。这种荧光归因于一种由咪唑酮环组成的生色团的存在，该咪唑酮环是由三肽-Ser65-Tyr66-Gly67-的翻译后修饰形成的。GFP已被广泛用作监测真核细胞和原核细胞中基因表达的报告蛋白，但关于产生荧光的化学机制却知之甚少。为了更好地理解这个问题，我们研究了带有生色团的GFP肽片段和生色团的合成模型化合物。结果表明，GFP生色团由咪唑酮环结构组成，发光体是生色团酚盐阴离子的单重激发态。此外，发光高度依赖于生色团周围的微环境，并且生色团外亚甲基双键异构化的抑制解释了其高效发光的原因。
