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PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate
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| Zeitschriftentitel: | Scientific Reports |
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| Personen und Körperschaften: | , , , , , , , , |
| In: | Scientific Reports, 7, 2017, 1 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Springer Science and Business Media LLC
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| Schlagwörter: |
| author_facet |
Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl |
|---|---|
| author |
Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl |
| spellingShingle |
Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl Scientific Reports PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate Multidisciplinary |
| author_sort |
lüddecke, jan |
| spelling |
Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl 2045-2322 Springer Science and Business Media LLC Multidisciplinary http://dx.doi.org/10.1038/s41598-017-01440-w <jats:title>Abstract</jats:title><jats:p>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<jats:sub>II</jats:sub> from <jats:italic>Synechococcus elongatus</jats:italic> 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 <jats:italic>in situ</jats:italic> glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in <jats:italic>Synechococcus elongatus</jats:italic> PCC 7942 cells and measured 2-OG concentrations in cell extracts from <jats:italic>Escherichia coli in vitro</jats:italic>. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.</jats:p> PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate Scientific Reports |
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| title |
PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_unstemmed |
PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_full |
PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_fullStr |
PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_full_unstemmed |
PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_short |
PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_sort |
pii protein-derived fret sensors for quantification and live-cell imaging of 2-oxoglutarate |
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Multidisciplinary |
| url |
http://dx.doi.org/10.1038/s41598-017-01440-w |
| publishDate |
2017 |
| physical |
|
| description |
<jats:title>Abstract</jats:title><jats:p>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<jats:sub>II</jats:sub> from <jats:italic>Synechococcus elongatus</jats:italic> 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 <jats:italic>in situ</jats:italic> glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in <jats:italic>Synechococcus elongatus</jats:italic> PCC 7942 cells and measured 2-OG concentrations in cell extracts from <jats:italic>Escherichia coli in vitro</jats:italic>. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.</jats:p> |
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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, Lüddecke, Jan, Francois, Liliana, Spät, Philipp, Watzer, Björn, Chilczuk, Tomasz, Poschet, Gernot, Hell, Rüdiger, Radlwimmer, Bernhard, Forchhammer, Karl |
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| description | <jats:title>Abstract</jats:title><jats:p>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<jats:sub>II</jats:sub> from <jats:italic>Synechococcus elongatus</jats:italic> 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 <jats:italic>in situ</jats:italic> glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in <jats:italic>Synechococcus elongatus</jats:italic> PCC 7942 cells and measured 2-OG concentrations in cell extracts from <jats:italic>Escherichia coli in vitro</jats:italic>. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.</jats:p> |
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| spelling | Lüddecke, Jan Francois, Liliana Spät, Philipp Watzer, Björn Chilczuk, Tomasz Poschet, Gernot Hell, Rüdiger Radlwimmer, Bernhard Forchhammer, Karl 2045-2322 Springer Science and Business Media LLC Multidisciplinary http://dx.doi.org/10.1038/s41598-017-01440-w <jats:title>Abstract</jats:title><jats:p>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<jats:sub>II</jats:sub> from <jats:italic>Synechococcus elongatus</jats:italic> 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 <jats:italic>in situ</jats:italic> glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in <jats:italic>Synechococcus elongatus</jats:italic> PCC 7942 cells and measured 2-OG concentrations in cell extracts from <jats:italic>Escherichia coli in vitro</jats:italic>. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.</jats:p> PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate Scientific Reports |
| spellingShingle | Lüddecke, Jan, Francois, Liliana, Spät, Philipp, Watzer, Björn, Chilczuk, Tomasz, Poschet, Gernot, Hell, Rüdiger, Radlwimmer, Bernhard, Forchhammer, Karl, Scientific Reports, PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate, Multidisciplinary |
| title | PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_full | PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_fullStr | PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_full_unstemmed | PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_short | PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| title_sort | pii protein-derived fret sensors for quantification and live-cell imaging of 2-oxoglutarate |
| title_unstemmed | PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate |
| topic | Multidisciplinary |
| url | http://dx.doi.org/10.1038/s41598-017-01440-w |