title: An adaptable luminescence resonance energy transfer assay for measuring and screening protein-protein interactions and their inhibition.
abstract: Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (∼ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA.
authors.forename: Engin
authors.initials: E
authors.lastname: Yapici
authors.forename: D Rajasekhar
authors.initials: DR
authors.lastname: Reddy
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Miller
mesh_terms: D004926:Escherichia coli / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006801:Humans; D015394:Molecular Structure; D011485:Protein Binding / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins; C058028:immunomycin; D013762:Tetrahydrofolate Dehydrogenase; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus