Duan Chenxi, Byrdin Martin, El Khatib Mariam, Henry Xavier, Adam Virgile, Bourgeois Dominique
Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France. CNRS, IBS, F-38044 Grenoble, France. CEA, IBS, F-38044 Grenoble, France.
Methods Appl Fluoresc. 2015 Jan 16;3(1):014004. doi: 10.1088/2050-6120/3/1/014004.
Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.
荧光蛋白特别容易发生光漂白，即由于发色团的光破坏导致荧光发射的永久性丧失。对于可逆转换荧光蛋白（RSFP）而言，它可以在非荧光状态和荧光状态之间来回切换，可实现的切换循环次数受到光漂白的限制，这一过程称为光疲劳。在许多基于RSFP重复光开关的先进应用中，尤其是在超分辨率荧光显微镜领域，光疲劳已成为一个关键限制因素。在此，基于我们之前对IrisFP（一种同样能够进行绿色到红色光转换的RSFP）光漂白机制的结构研究，我们展示了单突变体IrisFP-M159A的合理设计，其光稳定性显著增强。结果表明，在中等光照强度下，诸如EosFP、Dendra或Dronpa衍生物等类似IrisFP的珊瑚虫荧光蛋白的光漂白主要由氧依赖机制驱动，导致甲硫氨酸159发生不可逆的硫氧化。在不同的实验条件下，即在体外和细胞内，研究了IrisFP及其抗光漂白突变体IrisFP-M159A的光疲劳衰减曲线。尽管发现突变体的性能始终更优，但结果表明其切换行为强烈依赖于纳米环境。因此，一般来说，应在实际实验条件下评估RSFP的光稳定性和切换特性。
