Quantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor

Danai Laskaratou, Guillermo Solís Fernández, Quinten Coucke, Eduard Fron, Susana Rocha, Johan Hofkens, Jelle Hendrix and Hideaki Mizuno ()
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Danai Laskaratou: Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry
Guillermo Solís Fernández: Chem & Tech-Molecular Imaging and Photonics, Department of Chemistry
Quinten Coucke: Chem & Tech-Molecular Imaging and Photonics, Department of Chemistry
Eduard Fron: Chem & Tech-Molecular Imaging and Photonics, Department of Chemistry
Susana Rocha: Chem & Tech-Molecular Imaging and Photonics, Department of Chemistry
Johan Hofkens: Chem & Tech-Molecular Imaging and Photonics, Department of Chemistry
Jelle Hendrix: Chem & Tech-Molecular Imaging and Photonics, Department of Chemistry
Hideaki Mizuno: Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca2+ sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca2+. By monitoring the anisotropy, a Ca2+ rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET.

Date: 2021
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DOI: 10.1038/s41467-021-22816-7

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