Semi‐rational engineering of a coral fluorescent protein into an efficient highlighter

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62‐Tyr 63‐Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra‐) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green‐to‐red photoconvertibility. Substitution of the His 62‐Tyr 63‐Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi‐rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several‐fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two‐photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.