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Title: BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells
Authors: Yoshida, Tomoki
Kakizuka, Akira  kyouindb  KAKEN_id
Imamura, Hiromi  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0002-1896-0443 (unconfirmed)
Author's alias: 垣塚, 彰
今村, 博臣
Issue Date: 21-Dec-2016
Publisher: Springer Nature
Journal title: Scientific Reports
Volume: 6
Thesis number: 39618
Abstract: ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed "BTeam". BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the Îμ subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7-4.1 mM) and mitochondrial matrix (2.4-2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations.
Rights: © The Author(s) 2016. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
URI: http://hdl.handle.net/2433/217852
DOI(Published Version): 10.1038/srep39618
PubMed ID: 28000761
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