Loos Davey C, Habuchi Satoshi, Flors Cristina, Hotta Jun-Ichi, Wiedenmann Jörg, Nienhaus G Ulrich, Hofkens Johan
Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium.
J Am Chem Soc. 2006 May 17;128(19):6270-1. doi: 10.1021/ja0545113.
Proteins from the family of the green fluorescent protein (GFP) are presently extensively used in molecular and cellular biology. Recent studies suggest that isomerization of the chromophore occurs upon excitation and is involved in nonradiative deactivation. Using Raman spectroscopy, we report on photoinduced cis-trans isomerization in the red fluorescent protein eqFP611 from the sea anemone Entacmaea quadricolor. The crystal structure of eqFP611 shows that the chemical structure of the chromophore, p-hydroxybenzylidene-imidazolinone with an extended -conjugated system, is nearly identical to the chromophore of other red fluorescent proteins such as DsRed and HcRed. However, the chromophore of eqFP611 has a trans configuration whereas the chromophore of DsRed has a cis configuration. Upon irradiation with 532-nm light, the absorption of eqFP611 peaking at 559 nm diminished, and concomitantly a drastic decrease in the quantum yield of fluorescence as well as more complex decay kinetics was observed. Upon irradiation, changes in the Raman spectrum of eqFP611 were observed, and the relative intensities and peak positions of the irradiated eqFP611 showed striking similarity with the peaks in the Raman spectrum of DsRed. These observations are tentatively interpreted as trans-to-cis isomerization of the chromophore taking place upon irradiation together with the opening of new, nonradiative pathways.
绿色荧光蛋白（GFP）家族的蛋白质目前在分子和细胞生物学中得到广泛应用。最近的研究表明，发色团在激发时会发生异构化，并参与非辐射失活过程。利用拉曼光谱，我们报道了来自海葵Entacmaea quadricolor的红色荧光蛋白eqFP611中的光诱导顺反异构化。eqFP611的晶体结构表明，发色团的化学结构，即具有扩展共轭体系的对羟基苄叉咪唑啉酮，与其他红色荧光蛋白如DsRed和HcRed的发色团几乎相同。然而，eqFP611的发色团具有反式构型，而DsRed的发色团具有顺式构型。用532 nm光照射后，eqFP611在559 nm处的吸收峰减弱，同时观察到荧光量子产率急剧下降以及更复杂的衰减动力学。照射后，观察到eqFP611的拉曼光谱发生变化，照射后的eqFP611的相对强度和峰位与DsRed拉曼光谱中的峰表现出惊人的相似性。这些观察结果初步解释为照射时发色团发生反式到顺式异构化，同时开启了新的非辐射途径。
