Authors
Nivedita Singh, Smruti Ranjan Nayak, Sanjay Kumar Mohanty, Aishwarya Hanumantharaju, Amala Shaju, Mini Jose, Aravind Penmatsa, Deepak Nair
Published in
Advanced science (Weinheim, Baden-Wurttemberg, Germany). Pages e77693. Sep 27, 2026. Epub Sep 27, 2026.
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.
PMID:
42801590
Bibliographic data and abstract were imported from PubMed on 28 Sep 2026.
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