NMR samples are prepared in 20 mM KPi pH 7.0, 100 mM KCl, 4 mM βME, 0.5 mM EDTA, 0.05% NaN3, and 7% 2H2O. Protein concentration was 0.1–0.8 mM. NMR experiments were recorded on Agilent UNITY Inova 800 and 600 MHz NMR spectrometers and Bruker Avance III 600 and 700 MHz NMR spectrometers equipped with cryogenic probes. The experiments were run at 10 °C for isolated PhoA samples, and at temperatures ranging from 18 °C to 50 °C for the other samples. NMR spectra for the assignment and structure determination of the TF–PhoA complexes were recorded at 25 °C. Spectra were processed using the NMRPipe program (85 (link)) and data analysis was performed with Olivia (fermi.pharm.hokudai.ac.jp/olivia) and SPARKY software.
Binding affinities between TF and PhoA fragments were estimated using 1H-15N HSQC titration experiments, where unlabeled PhoA was titrated into labeled TF. Titration curves were obtained by plotting chemical shift perturbations (Δδppm) against the molar ratio of PhoA and TF. Non-linear least square fitting calculations were performed in GraphPad Prism (GraphPad Software, USA), using the following equation
where [L] and [P] are the concentrations of the peptide ligand (PhoA) and protein (TF), respectively, and Kd is the dissociation constant.
Binding affinities between TF and PhoA fragments were estimated using 1H-15N HSQC titration experiments, where unlabeled PhoA was titrated into labeled TF. Titration curves were obtained by plotting chemical shift perturbations (Δδppm) against the molar ratio of PhoA and TF. Non-linear least square fitting calculations were performed in GraphPad Prism (GraphPad Software, USA), using the following equation
where [L] and [P] are the concentrations of the peptide ligand (PhoA) and protein (TF), respectively, and Kd is the dissociation constant.