Reversible, Chemically Gated FRET via Ligand‐Activated Acceptors

ABSTRACT

Genetically encoded fluorogen‐activating tags enable conditional fluorescence, yet the structural and excited‐state mechanisms underlying ligand activation remain unclear. Here, we combine X‐ray crystallography, computational modelling, and fluorescence lifetime imaging microscopy (FLIM) to define the structural and photophysical basis of fluorogen activation in the Fluorescence‐Activating and Absorption‐Shifting Tag (FAST). Structures of apo and ligand‐bound FAST reveal the interface for fluorogen binding. In the apo state, N‐terminal residues occlude the pocket, whereas ligand binding displaces these elements to generate a solvent‐accessible cavity stabilized by ordered water molecules. Distinct hydroxybenzylidene rhodanine derivatives differentially tune interfacial geometry and excited‐state ensembles, producing single or multiexponential fluorescence lifetimes in living cells that primarily reflect modulation of excited‐state relaxation. Leveraging this ligand dependence, we utilize FAST as a chemically gated Förster resonance energy transfer (FRET) acceptor that induces reversible, concentration‐dependent donor lifetime shortening that does not require additional external controls. Using ligand‐controlled FLIM‐FRET, we resolve supramodular organization within Membrane‐Associated Guanylate Kinase (MAGUK) scaffolds in live cells, establishing FAST as a reversible photophysical module for mapping molecular architecture.