A single fluorescent protein-based sensor for in vivo 2-oxogluatarate detection in cell.
2-Oxoglutarate (2OG) is an important currency stands at the crossroad between carbon and nitrogen metabolism. Recent research found that 2OG acts as a signal in the regulation of nitrogen metabolism in prokaryote. While in eukaryotic cells, 2OG is also attractive since tricarboxylic acid cycle (TCA cycle) in tumor cells was found to undergo metabolic alterations such as the Warburg effect. A method of tracing this key metabolite 2OG at the cellular level is highly desirable. In order to visualize and monitor 2-oxoglutarate metabolism in single living cells, we developed a novel sensor by inserting the functional 2OG-binding domain GAF of the NifA protein into YFP. This sensor was found to be highly speciﬁc to 2OG. Following binding of 2OG, fluorescence intensity of the sensor increased with increasing 2OG concentration and reached a 1.5-fold maximum fluorescence signal change (F/F0-1), kinetics of fluorescence signal upon 2OG association with sensor was fast, the dynamic response range of the mOGsor sensors was 100 µM-100 mM. Dissociation between sensor and 2OG was verified both in vitro and in vivo. This sensor reported cellular 2OG dynamics in E. coli cells in real time upon different nutrition challenges and manifested the differences in 2OG pool accumulation and consumption rate.
Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
Chang
C
Zhang
Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China. Electronic address: bcye@ecust.edu.cn.
Bang-Ce
BC
Ye
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000032:analysis* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009584:Nitrogen / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D008164:Luminescent Proteins; C061338:NifA protein, Bacteria; D014157:Transcription Factors; C054562:yellow fluorescent protein, Bacteria; D009584:Nitrogen
2-Oxoglutarate; Genetically encoded biosensor; In vivo detection; Nitrogen metabolism