Autoitc isothermal titration calorimeter

The ITC experiments were performed using the AutoITC isothermal titration calorimeter (MicroCal Inc.) at 25°C. The procedure was performed according to Panuszko et al. (2009) (link). Briefly, lipoteichoic acid (LTA) from S. aureus or lipopolysaccharide (LPS) from P. aeruginosa 10 (both from SIGMA-Aldrich) and RAP-29 peptide were dissolved in 20 mM potassium phosphate buffer, pH 8.0, 10% glycerol. The RAP-29 peptide at concentration of 0.5 mM was gradually injected (29 injections for at 4-min intervals, 2 μL for the first injection only) into the reaction cell containing 0.05 mM solution of LTA or LPS. A background titration, consisting of the identical titrant solution but only the buffer solution in the sample cell, was subtracted from each experimental titration to account for heat of dilution. Mixing was carried out at constant 300 rpm. The standard procedure (CaCl₂ − EDTA titration) was performed to validate the apparatus and the stoichiometry, K, ΔH results were compared with those obtained for the test kit from Malvern Instruments Ltd. (Malvern, United Kingdom).

All ITC experiments were performed at 25°C using the AutoITC isothermal titration calorimeter (MicroCal Inc.). The measuring devices and experimental setup details were described previously (64 (link)). The reagents, P30 and LTA (Sigma-Aldrich catalog number L2515-5MG), were dissolved directly in 20 mM HEPES, pH 7.4. The experiment consisted of injecting 10.02 μL (29 injections, 2 μL for the first injection only) of 0.5 mM buffered solution of P30 into the reaction cell, which initially contained 0.05 mM buffered solution of LTA. A background titration, consisting of an identical titrant solution but with the buffer solution in the reaction cell only, was removed from each experimental titration. The LTA solution was injected at 4-min intervals. Each injection lasted 20 s. The stirrer speed was kept constant at 300 rpm. The CaCl₂-EDTA titration was performed to check the apparatus, and the results (stoichiometry, K, and ΔH) were compared with those obtained for the same samples (a test kit) at Malvern Instruments Ltd. (Malvern, UK).

All isothermal titration calorimetry (ITC) experiments were conducted in a 0.1 M sodium phosphate buffer (pH 6.8) at 25 °C using an AutoITC isothermal titration calorimeter (MicroCal, Malvern, UK), with 1.4491 mL of sample and reference cells. The cell containing deionized water was used as the reference. All solutions were degassed before titrations. The experiment consisted of injecting 10.02 μL (20 injections, 2 μL for the first injection only) of buffer solution of the appropriate antibiotic (1 mM) into the reaction cell initially containing xanthine in the buffer solution (15 mM). The titrant was injected in 5 min intervals to ensure that the titration peak returns to the baseline prior to the next injection. Each injection lasted 20 s. Background titrations were run using identical titrant with the pure buffer solution placed in the sample cell. To account for the heat of dilution, the result of a background titration was subtracted from each experimental titration. To achieve a homogeneous mixing in the cell, the stirrer speed was established at 300 rpm. To remove the effect of titrant diffusion across the syringe tip during the equilibration process, an initial 2 μL injection was removed from each data set before analysis. The data, specifically the heat normalized per mole of injectant, were processed with Origin 7 software from MicroCal.