Green fluorescent protein as a noninvasive intracellular pH indicator
Green fluorescent protein as a noninvasive intracellular pH indicator
复制标题
DOI：
10.1016/s0006-3495(98)77870-1
复制
发表时间：
1998-03-01
影响因子：
3.4
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Kneen, M;Farinas, J;Verkman, AS
研究发现，绿色荧光蛋白(GFP)突变体在水溶液和活细胞的胞内隔间中的吸光度和荧光强度强烈依赖于pH。PH滴定表明，重组GFP突变体的吸光度和荧光发生了10倍的可逆变化，pK(A)值分别为6.0(GFP-F64L/S65T)、5.9(S65T)、6.1(Y66H)和4.8(T2031)，Y66H的表观希尔系数为0.7，其他蛋白的表观希尔系数接近1。在pH为5-8的水溶液中，GFP-S65T的荧光光谱形状、寿命(2.8 ns)和圆二色光谱与pH无关，荧光对pH变化的响应可逆，但在较低pH时，质子化和构象都发生变化。为了评价GFP作为细胞内pH指示剂的作用，将针对GFP-F64L/S65T的cDNA导入CHO和LLC-PK1细胞的细胞质、线粒体、高尔基体和内质网。利用离子载体组合(黑素和CCCP)或洋地黄素，开发了校准程序来确定细胞内GFP荧光的pH依赖性。GFP-F64L/S65T在细胞质和细胞器中的pH敏感性与纯化的GFP-F64L/S65T在生理盐水中的pH敏感性相似。NH4Cl脉冲实验表明，胞内GFP荧光对pH变化的响应非常迅速。细胞内绿色荧光蛋白的应用包括细胞质和细胞器的pH测量、pH调节和线粒体pH对原载体的响应。这些结果确立了GFP作为细胞内pH的一个有针对性的、非侵入性的指示剂的应用。
It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pK(a) values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T2031) with apparent Hill coefficients of 0.7 for Y66H and similar to 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in 5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.