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 (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 (>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.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions

“…Another important component of the sensor design is the eDHFR domain that binds to TMP-linked small molecules with high affinity ( K D , ∼1 nM) and selectivity ( Miller et al., 2005 ). In our prior work, we have shown that TMP-coupled Tb(III) complexes bind effectively to eDHFR fusion proteins in vitro ( Rajapakse et al., 2009 ), in lysates ( Yapici et al., 2012 ) and in living mammalian cells ( Rajapakse et al., 2010 ). Given the high affinities of TMP for eDHFR and of commonly used chelators for Tb(III) ( K A > 10 14 M −1 ) ( Selvin, 1996 ), assemblies of eDHFR and TMP-Tb(III) remain stable in cells or at high dilution in challenging environments.…”

Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

“…In an in vitro assay performed in 96-well plates, we titrated a fusion of GFP to FK506 binding protein 12 (GFP–FKBP12) against a fixed concentration of eDHFR fused to the rapamycin binding domain of mTOR (FRB–eDHFR). Following the addition of rapamycin (to induce protein interaction) and the TMP-Tb(III) complex 7 , we observed a ∼3500% increase in the long-lifetime, Tb-to-GFP-sensitized emission . The high dynamic range of Tb(III)-based FRET (>30-fold) greatly exceeds the best dynamic range for nondimerizing fluorescent proteins (<5-fold) observed in vitro and hints at the potential sensitivity of lanthanide-based FRET microscopy. , …”

“…The rapamycin-induced interaction between FKBP12 and FRB , presents an ideal model system for quantifying the detection limit, SNR, dynamic range, and other measures of the image quality that may be observed with time-gated FRET imaging of protein–protein interactions. Because both proteins diffuse throughout the cytoplasm and nucleus when expressed in mammalian cells, cytoplasmic signal levels are proportional to the total cellular protein concentration.…”

“…Enzymes and cloning reagents were obtained from New England Biolabs, Inc. Cell culture reagents were purchased from Life Technologies, Inc. Plasmid vectors pRSETB–EGFP–FKBP12 and pRSETB–FRB–eDHFR were described previously . MDCKII cells were obtained from the American Type Culture Collection (cat.…”

Efficient route to pre-organized and linear polyaminopolycarboxylates: Cy-TTHA, Cy-DTPA and mono/di- reactive, tert -butyl protected TTHA/Cy-TTHA

“…Spurred by our interest in developing luminescent lanthanide complexes for bioanalytical and imaging applications, 10,11,60–63 we sought to develop more efficient strategies for preparing Cy-TTHA ( 6 ) and Cy-DTPA ( 11 ) derivatives. Herein, we report a modular, five-step syntheses of Cy-TTHA ( 6 ) and Cy-DTPA ( 11 ) with good overall yields.…”