title: Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.
abstract: 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.
authors.forename: Kalin V
authors.initials: KV
authors.lastname: Vasilev
authors.forename: Eugenio
authors.initials: E
authors.lastname: Gallo
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Shank
authors.affiliation: Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, Pennsylvania 15213, USA. jarvik@cmu.edu.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Jarvik
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002232:Carbocyanines; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D008024:Ligands; D008565:Membrane Proteins / Q000032:analysis*; D008722:Methods; D060066:Protein Interaction Maps; D055503:Protein Multimerization
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D007211:Indoles; D008024:Ligands; D008565:Membrane Proteins; C572069:dimethylindole red