Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP

The site-directed mutagenesis of the monomeric red fluorescent protein TagRFP and its variants was performed with the goal of generating reversibly photoactivatable fluorescent proteins. Amino acids at positions 69, 148, 165, 179, and 181 (enumeration according to the green fluorescent protein GFP) were shown to play a key role in the manifestation of the photoactivatable properties. A reversibly photoactivatable red fluorescent protein KFP-HC with excitation and emission maxima at 585 and 615 nm, respectively, was generated. The KFP-HC fluorescent intensity was decreased by 5–10 times under green light (530–560 nm) irradiation (due to the fall of the fluorescence quantum yield) and restored under irradiation with blue light (450–490 nm) or after incubation in the dark (recovery half-time of 30 min).