IIT@NEST, Center for Nanotechnology Innovation, Piazza San Silvestro 12, I-56127 Pisa, Italy.
J Am Chem Soc. 2010 Jan 13;132(1):85-95. doi: 10.1021/ja9014953.
Reversibly photoswitchable (i.e., photochromic) fluorescent proteins open the way to a number of advanced bioimaging techniques applicable to living-cell studies such as sequential photolabeling of distinct cellular regions, innovative FRET schemes, or nanoscopy. Owing to the relevance of fluorescent proteins from Aequorea victoria (AFPs) for cell biology, a photochromic "toolbox" constituted by several AFPs is highly desirable. Here we introduce four new photochromic AFPs whose reversible photoswitching occurs between the native bright and a dark state at low illumination power, on account of a very efficient cis-trans photoisomerization. Most remarkably, the optical bistability of these AFPs derives from the single E222Q mutation in the primary sequence. Apparently, the E222Q substitution can restore the intrinsic photochromic behavior of the isolated chromophore. The significance of these mutants for high-resolution in vivo cell imaging is shown by means of photochromic FRET experiments.
可光致变色（即光致变色）荧光蛋白为许多先进的生物成像技术开辟了道路，这些技术可应用于活细胞研究，例如对不同细胞区域进行顺序光标记、创新的 FRET 方案或纳米显微镜技术。由于维多利亚水母（AFPs）的荧光蛋白与细胞生物学相关，因此非常需要由几种 AFP 组成的光致变色“工具包”。在这里，我们介绍了四个新的光致变色 AFP，它们在低光照功率下在天然亮态和暗态之间发生可逆光开关，这是由于非常有效的顺反异构化。最显著的是，这些 AFP 的光学双稳性源于其一级序列中的单个 E222Q 突变。显然，E222Q 取代可以恢复分离的生色团的固有光致变色行为。通过光致变色 FRET 实验证明了这些突变体在高分辨率体内细胞成像中的重要性。
