Bell A F, He X, Wachter R M, Tonge P J
Department of Chemistry, Graduate Program in Biophysics, and Graduate Program in Molecular and Cellular Biochemistry, SUNY at Stony Brook, Stony Brook, New York 11794-3400, USA.
Biochemistry. 2000 Apr 18;39(15):4423-31. doi: 10.1021/bi992675o.
We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).
我们展示了使用752 nm激发光获得的野生型绿色荧光蛋白（GFP）及其S65T突变体的拉曼光谱，同时还展示了一种模型发色团——4-(4-羟苯基)亚甲基-2-甲基-5-氧代咪唑乙酸乙酯的数据。在pH值1 - 14范围内，该模型化合物有两个宏观pK(a)值，分别为1.8和8.2，分别归因于咪唑啉酮环氮和酚羟基的电离。将模型发色团与野生型GFP和S65T突变体中的发色团进行比较发现，在这些特定的GFP蛋白中不存在咪唑啉酮环氮和酚氧均质子化的阳离子形式。我们的结果没有提供任何证据表明发色团的两性离子形式（酚基团去质子化且咪唑啉酮环氮质子化）存在于GFP蛋白中。此外，由于拉曼带的位置是基态结构特有的性质，这些数据使我们能够研究蛋白质 - 发色团相互作用如何影响发色团的基态结构，而不受激发态效应的影响。结果发现，与荧光特性最相关的发色团阴离子形式的基态结构强烈依赖于发色团环境，而中性形式似乎不敏感。通过绘制吸收最大值与在1610 - 1655 cm(-1)范围内发现的拉曼带波数的关系图，证明了吸收特性与基态结构之间的线性相关性。
