Structural and spectral response of green fluorescent protein variants to changes in pH
Structural and spectral response of green fluorescent protein variants to changes in pH
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DOI：
10.1021/bi9902182
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发表时间：
1999-04-27
期刊：
影响因子：
2.9
通讯作者：
Remington, SJ
中科院分区：
文献类型：
--
作者：
Elsliger, MA;Wachter, RM;Remington, SJ
维多利亚水母的绿色荧光蛋白（GFP）已成为分子和细胞生物学的有用工具。最近，已经发现，大多数GFP突变体的荧光光谱响应迅速，可逆的pH值变化，使它们有用的细胞内pH值的探针，探索流行的GFP S65 T变体的滴定行为的结构基础，我们确定了高分辨率的晶体结构在pH值8.0和4.6。的结构揭示了与发色团的氢键模式的变化，这表明pH敏感性来自发色团酚盐的质子化。在黄色荧光蛋白（SG 5G/V68 L/S72 A/T203 Y）中设计突变以改变溶剂可及性（H148 G）并修饰发色团附近的极性基团（H148 Q，E222 Q）。这些变体的pH滴定表明，发色团pK（a）可以在6至8的宽范围内调节，从而允许在pH 5至pH 9的范围内进行pH测定。最后，使用诱变将pK（a）从6.0（S65 T）提高至7.8（S65 T/H148 D）。与其他变体不同，S65 T/H148 D表现出两个绿色荧光的pH依赖性激发峰，具有干净的等吸光点。这就提出了在这个等吸光点上使用荧光作为内部参考的有趣的可能性。提出了在细胞和发育生物学中的实际真实的实时体内应用。
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH, To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (SG5G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pK(a) can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9, Finally, mutagenesis was used to raise the pK(a) from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point, as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.