title: BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells.
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 ε 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.
authors.affiliation: Department of Functional Biology, Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Department of Functional Biology, Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kakizuka
authors.affiliation: Department of Functional Biology, Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis*; D001426:Bacterial Proteins / Q000737:chemistry; D015374:Biosensing Techniques; D003593:Cytoplasm / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D000255:Adenosine Triphosphate