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P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse bi...

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Autores principales: Lüddecke, Jan, Francois, Liliana, Spät, Philipp, Watzer, Björn, Chilczuk, Tomasz, Poschet, Gernot, Hell, Rüdiger, Radlwimmer, Bernhard, Forchhammer, Karl
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431102/
https://www.ncbi.nlm.nih.gov/pubmed/28469248
http://dx.doi.org/10.1038/s41598-017-01440-w
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author Lüddecke, Jan
Francois, Liliana
Spät, Philipp
Watzer, Björn
Chilczuk, Tomasz
Poschet, Gernot
Hell, Rüdiger
Radlwimmer, Bernhard
Forchhammer, Karl
author_facet Lüddecke, Jan
Francois, Liliana
Spät, Philipp
Watzer, Björn
Chilczuk, Tomasz
Poschet, Gernot
Hell, Rüdiger
Radlwimmer, Bernhard
Forchhammer, Karl
author_sort Lüddecke, Jan
collection PubMed
description The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can’t be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein P(II) from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.
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spelling pubmed-54311022017-05-16 P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl Sci Rep Article The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can’t be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein P(II) from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening. Nature Publishing Group UK 2017-05-03 /pmc/articles/PMC5431102/ /pubmed/28469248 http://dx.doi.org/10.1038/s41598-017-01440-w Text en © The Author(s) 2017 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/.
spellingShingle Article
Lüddecke, Jan
Francois, Liliana
Spät, Philipp
Watzer, Björn
Chilczuk, Tomasz
Poschet, Gernot
Hell, Rüdiger
Radlwimmer, Bernhard
Forchhammer, Karl
P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_full P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_fullStr P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_full_unstemmed P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_short P(II) Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
title_sort p(ii) protein-derived fret sensors for quantification and live-cell imaging of 2-oxoglutarate
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431102/
https://www.ncbi.nlm.nih.gov/pubmed/28469248
http://dx.doi.org/10.1038/s41598-017-01440-w
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