We describe a novel biosensor system for reporting proximity between cell surface proteins
in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating
proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen
molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins
were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd-
FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP.
Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR)
consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the
fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby
brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the
new proximity assay TEFLA, for tethered fluorogen assay.
Keywords: Membrane proteins, protein interaction, protein proximity, biosensor, fluorogen-activating protein, tethered
fluorogen assay, TEFLA.
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