Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

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. This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.