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An Adaptable Luminescence Resonance Energy Transfer Assay for Measuring and Screening Protein–Protein Interactions and their Inhibition.
Author(s) -
Yapici Engin,
Reddy D. Rajasekhar,
Miller Lawrence W.
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100710
Subject(s) - fkbp , förster resonance energy transfer , dihydrofolate reductase , chemistry , green fluorescent protein , protein–protein interaction , biophysics , binding protein , target protein , biochemistry , lysis , plasma protein binding , fluorescence , biology , enzyme , physics , quantum mechanics , gene
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.