Microcal peaq itc isothermal titration calorimeter

Isothermal titration calorimetry (ITC) was used to determine the equilibrium dissociation constants (K_D) and binding stoichiometries (n) of the different ObgE proteins (wild type or mutant) for GDP, ppGpp, and GTPγS. The ITC experiments were performed at 25 °C on a MicroCal PEAQ-ITC isothermal titration calorimeter (Malvern Panalytical) using a reference power of 10 µcal/s. Depending on the affinity, a protein concentration between 50 and 90 µM and a ligand concentration between 525 µM and 4.5 mM were used in the cell and syringe, respectively. All measurements were performed in a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, and 1 mM β-mercaptoethanol. During each ITC experiment, 18 injections of 2 µL were performed, preceded by a test injection of 0.5 µL. To determine the K_D-values and binding stoichiometries, the obtained data were fit on the single binding site model provided by the MicroCal PEAQ-ITC analysis software. In case of the ObgE_T174I protein, which displayed low binding affinities for all tested nucleotides, only the K_D-value could be derived from the fit, while the binding stoichiometry was fixed at 1.

ITC experiments were carried in a MicroCal PEAQ-ITC isothermal titration calorimeter (Malvern Panalytical)^{22 (link)}. Briefly, complex 1a and recombinant VBC complex were dialyzed into the ITC buffer (20 mM Bis–Tris, 150 mM NaCl, 2 mM DTT, 1% DMSO) overnight. Complex 1a (300 μM) was titrated against 30 μM VBC complex, consisting of 19 injections of 2 μL complex 1a solution at a rate of 2 s/μL at 150 s time intervals. An initial injection of ligand (0.4 μL) was made and discarded during data analysis. The experiment was carried out at 25 °C while stirring at 750 rpm. The generated data were fitted to a single binding site model using the Setup MicroCal PEAQ-ITC Analysis Software provided by the manufacturer. Three control titrations, in which (1) 1a is titrated into VBC buffer; (2) VBC buffer is titrated into VBC complex; (3) VBC buffer is titrated into VBC buffer, were also analyzed by using a composite model.