2-oxoglutarate (2-OG) is a key intermediate in the central metabolism, and locates at the junction between the Krebs cycle and assimilatory pathway of nitrogen. This central carbon intermediate served as sole carbon skeleton for the assimilation of nitrogen. 2-OG not only plays a critical role in metabolism, but also acts as a signaling molecule in a variety of organisms.Tracing intracellular level of 2-oxoglutarate in real-time would show great significance in related studies of cell metabolism and signal transduction. Recently, we develop a novel genetically encoded 2-OG sensor based on fluorescent resonance energy transfer (FRET) by inserting a functional 2-OG-binding domain GAF of NifA protein from Azotobacter vinelandii between the FRET pair YFP/CFP, and demonstrated the feasibility of this sensor for the visualization of 2-OG both in vitro and in vivo. We optimized the peptide lengths of the binding domain. The resulting sensor OGsor-G9 exhibited a maximal average ratio change ?R of 0.95, a 6-fold improvement over the 0.16 of OGsor-G, and showed high selectivity to 2OG over other related compounds. The dynamic response range of the OGsor-G9 is 100 �M-10 mM, covering a wide range of physiologically relevant 2OG concentrations. We also attained a mono-fluorescent sensor mOGsor by inserting the functional domain GAF into YFP protein. Following binding of 2OG, fluorescence intensity of the sensor increased with increasing 2OG concentration and reached a 1.5-fold maximum fluorescence signal change, kinetics of fluorescence signal upon 2OG association with sensor was fast. Our results illustrated that the two OGsor sensors are a powerful tool for scrutinizing the signal transdution and cross-talk mechanism of central carbon metabolism and nitrogen metabolism responding the nutritional states in cell.

Quantitative monitoring of 2-oxoglutaratein cells by genetically-encoded biosensors

247th National Meeting (2014)

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