45SD

FAST_apo_crystal

Summary for 45SD

• Entry DOI: 10.2210/pdb45sd/pdb
• Related: 7AV6
• Descriptor: Fluorescence-Activating and Absorption-Shifting Tag (FAST) (2 entities in total)
• Functional Keywords: fluorescent protein, fret acceptor, ligand dependent fluorescence
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 13603.39

Authors

Nayak, S.R.,Singh, N.,Penmatsa, A.,Nair, D.K. (deposition date: 2026-09-08, release date: 2026-10-28)

Primary citation

Singh, N.,Nayak, S.R.,Mohanty, S.K.,Hanumantharaju, A.,Shaju, A.,Jose, M.,Penmatsa, A.,Nair, D.
Reversible, Chemically Gated FRET via Ligand-Activated Acceptors.
Adv Sci, :e77693-e77693, 2026

PubMed 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.

PubMed: 42801590
DOI: 10.1002/advs.77693

Experimental method

X-RAY DIFFRACTION (2.9 Å)