Fig. 3: Binding affinities of nucleotides to KtrA and nucleotide-dependent protein stabilization.
KtrA was purified via SEC and the affinities of nucleotides were determined using ITC. a The upper panel shows the raw heat exchange data of ATP and ADP binding to KtrA, respectively. The lower panels present the integrated injection heat pulses, normalized per mole of injection. The binding curves were fitted by a one-site binding model, resulting in the indicated dissociation constants (KD). Respective KD values and SDs are noted for triplicates of each titration. b, c Thermostability of KtrA was investigated by differential scanning fluorimetry. Purified KtrA was mixed with different concentrations of ATP (red) or ADP (blue) and changes in SyproOrange fluorescence (λex 470 nm; λem 555 nm) were detected over a temperature increase from 25 to 80 °C. The peak of the raw data's first derivative reflects the protein's melting point. d Melting temperatures of purified KtrA (black) and in the presence of different concentrations of ATP (red) or ADP (blue) (10 µM to 1000 µM). ADP binding induces higher thermal stability at significantly lower concentrations. e To test ATP/ADP displacement, competitive binding experiments were performed. 5 µM KtrA were pre-incubated with either 500 µM ATP (black-framed blue point) or 500 µM ADP (black-framed red point) and supplied with different concentrations of ADP (blue) or ATP (red), respectively. Displacement of both nucleotides was observed. Data points in d and e show the means and SDs of technical triplicates.
