Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions
Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions
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DOI：
10.1007/s10895-012-1145-y
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发表时间：
2013-03-01
影响因子：
2.7
通讯作者：
Abraham, Beata
中科院分区：
文献类型：
--
作者：
Balint, Emese-Eva;Petres, Judit;Abraham, Beata
通过位点定向诱变将两种组氨酸引入增强型绿色荧光蛋白的结构中，取代202位的丝氨酸和204位的谷氨酰胺，从而通过在发色团附近创建可能的金属结合位点来提高蛋白质对不同金属离子的敏感性。两种蛋白质（野生型和双组氨酸突变体）的吸光度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℃）下测量金属离子浓度的荧光猝灭。在最佳条件下（pH = 7.5, 20℃），突变体的K-d为16 nM，与铜的结合强度是野生型EGFP的200多倍。
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 A degrees C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 A degrees C) the mutant's K-d is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.