Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, Wageningen, The Netherlands.
PLoS One. 2010 Nov 5;5(11):e13862. doi: 10.1371/journal.pone.0013862.
Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.
最近，我们描述了 ATP 会引起荧光共振能量转移（FRET）传感器的 YFP/CFP 荧光强度变化，这些传感器基于 CFP-YFP。为了深入了解这一现象，我们采用荧光寿命光谱法更详细地分析了 ATP 对这些荧光蛋白的影响。使用不同的供体和受体对，我们发现 ATP 仅影响基于 CFP-YFP 的版本。对所使用蛋白的纯化单体的后续分析表明，ATP 对 CFP 的荧光寿命特性有直接影响。由于 CFP 的荧光寿命分析受到不同寿命的存在而变得相当复杂，我们测试了 CFP 的一种变体，即 Cerulean，作为单体和在我们的 FRET 构建体中。令人惊讶的是，这种 CFP 变体在荧光寿命中没有表现出与 ATP 浓度相关的变化。CFP 和 Cerulean 之间最重要的区别是在位置 148 存在一个组氨酸残基。事实上，将 CFP 中的这个组氨酸突变为天冬氨酸，会导致与 Cerulean 荧光 FRET 传感器观察到的相同的荧光特性。因此，我们得出结论，CFP 荧光寿命的变化受 ATP 负电荷与位置 148 的正电荷之间可能的静电相互作用的特异性影响。显然，需要进一步的物理化学特性分析来解释 CFP 荧光特性对环境（即 ATP 浓度）条件变化的敏感性。
