Prism 5 program

All experiments were performed at least in triplicates. Data were analyzed using the Prism 5 Program (Graph Pad, San Diego, CA, USA). Values are presented with individual data points + mean. Statistical difference was determined by one-way ANOVA followed by Tukey’s range test. A P-value of <0.05 was regarded statistically significant.

E. coli BL21(DE3) cells carrying pET derivative plasmids were grown at 37 °C in Lysogeny Broth to an OD₆₀₀ of 0.5, and gene expression was induced by the addition of 100 µM IPTG for 12 h at 17 °C. Proteins were purified by immobilized metal ion chromatography as previously described [35 (link)]. Spectroscopic analyses were monitored in 96-well plates using a TECAN Infinite^®® 200 series microplate reader (Hännedorf, Switzerland) equipped with a dual injector head for kinetic measurements. The excitation and emission bandwidths of the instrument are 9 nm and 20 nm, respectively. For in vitro and in vivo experiments, fluorescence emissions were recorded using an excitation at 425 nm, and emissions at 530 nm and 480 nm for YFP and CFP, respectively. The FRET ratio was calculated as the 530/480 intensity ratio (YFPem/CFPem) and reflects the level of excitation of YFP by the CFP, which is directly linked to the distance between the two fluorophores. The relative FRET ratio was calculated as the FRET ratio for a specific 2-OG concentration relative to the same ratio without 2-OG. The data were analyzed by the PRISM 5 program (GraphPad Software, San Diego, CA, USA). For in vitro experiments, fluorescence emissions were recorded 5 min after ligand addition.