Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP
Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP
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DOI：
10.1007/s00216-012-5911-0
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发表时间：
2012-05-01
影响因子：
4.3
通讯作者：
Schleifenbaum, Frank
中科院分区：
文献类型：
--
作者：
Wierer, Sebastian;Peter, Sebastien;Schleifenbaum, Frank
为了定量分析活的(植物)细胞中的分子过程，如对环境信号和内源信号的感知和处理，需要在光学和光谱技术方面采用新的组合方法，并且部分已经在生命科学的许多领域建立。活体细胞生物学过程分析的一个特点是使用可见的荧光蛋白来产生荧光融合蛋白。最近在产生氧化还原敏感的绿色荧光蛋白突变体(RoGFP)方面取得了进展，该突变体的光谱性质随着周围介质氧化还原状态的变化而发生变化。一种已建立的使用roGFP探测局部氧化还原电位的方法是基于比率计算法。这种读出方式需要两个激发波长，这使得该技术不太适合于例如动态样本的活体研究。我们通过从头计算阐明了roGFP氧化还原敏感性的来源，揭示了生色团的质子化平衡随氧化还原势的变化。基于这一发现，我们测试和比较了单波长激发下不同的光谱读出模式，以确定局部氧化还原电位，并将这些技术应用于活细胞分析。
For the quantitative analysis of molecular processes in living (plant) cells, such as the perception and processing of environmental and endogenous signals, new combinatorial approaches in optical and spectroscopic technologies are required and partly already became established in many fields of the life sciences. One hallmark of the in vivo analysis of cell biological processes is the use of visible fluorescent proteins to create fluorescent fusion proteins. Recent progress has been made in generating a redox-sensitive mutant of green fluorescent proteins (roGFP), which exhibits alterations in its spectral properties in response to changes in the redox state of the surrounding medium. An established method to probe the local redox potential using roGFP is based on a ratiometric protocol. This readout modality requires two excitation wavelengths, which makes the technique less suited for in vivo studies of e.g. dynamic samples. We clarify the origin of the redox sensitivity of roGFP by ab initio calculations, which reveal a changed protonation equilibrium of the chromophore in dependence on the redox potential. Based on this finding, we test and compare different spectroscopic readout modalities with single wavelength excitation to determine the local redox potential and apply these techniques to live cell analytics.