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

• Published in: iScience Vol. 23; no. 9; p. 101533
• Main Authors: Chen, Ting, Pham, Ha, Mohamadi, Ali, Miller, Lawrence W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-09-2020; Elsevier
• Subjects: Biomolecular Engineering; Molecular Interaction; Molecular Spectroscopy Techniques; Sensor; Molecular Spectroscopy Techniques; Molecular Interaction; Biomolecular Engineering; Sensor
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2020.101533

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.
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•Non-invasive, microscopic imaging or screening of protein-protein interactions•Intracellular assembly of sensor polypeptides with luminescent Tb(III) complexes•High dynamic range with time-gated detection of Tb(III)-to-GFP sensitized emission
Sensor; Molecular Spectroscopy Techniques; Molecular Interaction; Biomolecular Engineering