Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions
journal contributionposted on 2021-12-17, 16:31 authored by T Chen, H Pham, A Mohamadi, Lawrence MillerLawrence Miller
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.
Targeted Lanthanide Contrast Agents for in Cellulo Single Molecule Imaging | Funder: National Institutes of Health (National Institute of General Medical Sciences) | Grant ID: R01GM081030
Multiplexed FRET Imaging of Protein Interactions with Lanthanide Probes (EQUIPMENT SUPPLEMENT) | Funder: National Institutes of Health (National Institute of General Medical Sciences) | Grant ID: R01GM081030
CitationChen, T., Pham, H., Mohamadi, A.Miller, L. W. (2020). Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions. iScience, 23(9), 101533-. https://doi.org/10.1016/j.isci.2020.101533