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Title: | Quantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor | Authors: | Laskaratou, Danai Fernández, Guillermo Solís Coucke, Quinten Fron, Eduard Rocha, Susana Hofkens, Johan HENDRIX, Jelle Mizuno, Hideaki |
Issue Date: | 2021 | Publisher: | NATURE RESEARCH | Source: | Nature Communications, 12 (1) (Art N° 2541) | 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. | Document URI: | http://hdl.handle.net/1942/34098 | e-ISSN: | 2041-1723 | DOI: | 10.1038/s41467-021-22816-7 | ISI #: | 000656480900008 | Rights: | The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. | Category: | A1 | Type: | Journal Contribution | Validations: | ecoom 2022 |
Appears in Collections: | Research publications |
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s41467-021-22816-7.pdf | Published version | 2.71 MB | Adobe PDF | View/Open |
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