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

ABSTRACT: 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.

SUBMITTER: Chen T 

PROVIDER: S-EPMC7509216 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Chen Ting T   Pham Ha H   Mohamadi Ali A   Miller Lawrence W LW  

iScience 20200906 9

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  ...[more]