Single amino acid replacement makes Aequorea victoria fluorescent proteins reversibly photoswitchable.

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.