Department of Inorganic Substances Technology and Environment Protection, Politechnica University of Bucharest, Polizu street No. 1-7, 011061 Bucharest, Romania.
J Fluoresc. 2013 Mar;23(2):273-81. doi: 10.1007/s10895-012-1145-y. Epub 2012 Nov 6.
Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45 °C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 °C) the mutant's Kd is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.
通过定点突变，将两个组氨酸引入增强型绿色荧光蛋白的结构中，取代位置 202 的丝氨酸和位置 204 的谷氨酰胺，通过在生色团附近创建可能的金属结合位点，提高蛋白质对不同金属离子的灵敏度。两种蛋白质（野生型和双组氨酸突变体）的吸收光谱和荧光光谱没有明显差异，表明突变不会改变荧光团的环境。在不同 pH 值（6.5-8）和温度（20-45°C）下，通过改变金属离子浓度来测量荧光猝灭。在最佳条件下（pH=7.5，20°C），突变体的 Kd 值为 16 nM，它与铜的结合强度比野生型 EGFP 强 200 多倍。
