Fluorogenesis: Inducing Fluorescence in a Non-Fluorescent Protein Through Photoinduced Chromophore Transfer of a Genetically Encoded Chromophore

ABSTRACT Fluorescent proteins, while essential for bioimaging, are limited to visualizing cellular localization without offering additional functionality. We report for the first time a strategy to expand the chemical, structural, and functional diversity of fluorescent proteins by harnessing light to induce red fluorescence in a previously non-fluorescent protein. We accomplish this by inducing the transfer of the genetically encoded chromophore from a photocleavable protein (PhoCl1) to a non-fluorescent kinase ( Mj RibK) inducing red fluorescence in the latter. We have employed analytical and spectroscopic techniques to validate the presence of red fluorescence in Mj RibK. Furthermore, molecular dynamics simulations were carried out to investigate the amino acid residues of Mj RibK involved in the generation of red fluorescence. Finally, we demonstrate the ability of the red fluorescent Mj RibK to operate as a cyclable high-temperature sensor. We anticipate that this light-induced chromophore transfer strategy will open new possibilities for developing multifunctional genetically encoded fluorescent sensors.