pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins
pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins
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DOI：
10.1007/s00216-015-8636-z
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发表时间：
2015-05-01
影响因子：
4.3
通讯作者：
Merola, Fabienne
中科院分区：
文献类型：
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作者：
Betolngar, Dahdjim-Benoit;Erard, Marie;Merola, Fabienne
一般认为，基因编码报告者携带的常见的青色和黄色荧光蛋白对接近生理pH范围的pH值具有很强的敏感性。我们研究了这些pH响应对模型福斯特共振能量传递（FRET）串联和基于FRET的camp依赖性蛋白激酶活性（AKAR）在活BHK细胞胞浆中表达的细胞内信号的影响，同时通过尼日利亚菌素离子载体改变细胞内pH。虽然在某些情况下，供体和受体同时的pH敏感性可能会相互掩盖，但与AKAR生物传感器的功能响应相比，扰动的幅度可能非常显著。用光谱上相同但对pH不敏感的海蓝宝石变体（pK(1/2) = 3.3）代替CFP供体，极大地改变了生物传感器的pH响应，并使黄色受体能够进行酸性转变。我们建立了一个简单的ph依赖的FRET模型，并用它来描述预期的ph诱导的荧光寿命和比率信号的变化。该模型定性地解释了大部分观察结果，但揭示了细胞质AKAR生物传感器在酸性ph值处的复杂行为，与额外的FRET贡献有关。这项研究强调了pH变化对活细胞中FRET报告信号的主要和复杂的影响。
It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Forster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK(1/2) = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.