Design and Evolution of a Phosphorescent Protein via the Proximal Encoding of Lanthanide and the Antenna Chromophore

Genetically encoded phosphorescent proteins with extended luminescence lifetimes provide an orthogonal channel for biological imaging and detection. While conventional fluorescent proteins typically exhibit nanosecond-scale lifetimes, the development of proteins with longer lifetimes enables time-resolved detection and enhanced signal-to-noise ratios. Here, we designed a novel phosphorescent protein system by incorporating photosensitizing unnatural amino acids (UAAs) proximal to the metal center of a lanthanide binding protein (LanM). Through systematic optimization of the incorporation sites, we achieved considerable enhancement in lanthanide phosphorescence compared with that of the wild-type LanM protein. The subsequent directed evolution of LanM and chemical evolution of UAA yielded variants with an additional muti-fold increase in signal intensity. This iterative optimization strategy generated phosphorescent proteins with extended lifetimes of up to 500 μs and significantly increased brightness. Using this phosphorescence protein platform, a europium sensor with a signal-to-noise ratio of more than 100 for 200 nM Eu(III) and a detection limit of less than 10 nM was developed. In addition, a protease sensor was further designed by inserting a cleavage site into a loop of the phosphorescent protein, achieving remarkable signal-to-noise ratios at nanomolar concentrations. Finally, this phosphorescent protein was fused to the affibody and further used for immunofluorescence imaging. These applications demonstrated a novel platform for developing genetically encoded sensors with enhanced detection sensitivity through time-resolved measurements.