Significance: We developed a small protein tag enabling fluorescent labeling of proteins in living cells and in multicellular organisms through the specific binding and activation of a cell-permeant and nontoxic fluorogenic ligand. This tag, called Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), was engineered by directed evolution from the Photoactive Yellow Protein. Y-FAST distinguishes itself from other labeling methods because the fluorogen binding is highly dynamic and fully reversible. Apart from providing new opportunities in superresolution imaging and biosensor design, this feature enables rapid switching on and off of the fluorescence of a fusion protein by addition or withdrawing of the fluorogenic ligand, opening exciting ways to perform sequential multiplexing imaging.

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescence-Activating and absorption-Shifting Tag), which specifically and reversibly binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs, splitFAST displays rapid and reversible complementation, allowing the real-time visualization of both the formation and the dissociation of a protein assembly.