PII 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...
Gespeichert in:
Veröffentlicht in: | Scientific reports 2017-05, Vol.7 (1), p.1-13, Article 1437 |
---|---|
Hauptverfasser: | , , , , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | 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. |
---|---|
ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/s41598-017-01440-w |