Thermodynamics of LPS Binding to Thanatin

Microcalorimetric measurements of the binding of thanatin or divalent cations (Mg²⁺, Ca²⁺) to LPS were performed on a MicroCal Auto-ITC200 instrument (Malvern Instruments, Malvern, UK)^{35 (link)}. LPS (E. coli serotype 055:B5, Sigma, USA) was dissolved in 20 mM Tris-HCl (pH = 6.8) or 10 mM PBS (pH = 7.4), vortexed vigorously for 15 min, and sonicated for 15 min at 60 °C. The LPS solution was sonicated for 5 min prior to use. Thanatin was dissolved in Tris-HCl (pH = 6.8) and titrated into LPS in Tris-HCl (pH = 6.8). Divalent cations (Mg²⁺, Ca²⁺) were dissolved in PBS (pH = 7.4) and titrated into LPS in PBS (pH = 7.4). All samples were degassed for 10 min in a sonication bath before the experiments. These experiments were performed at 25 °C. The generated peaks were integrated using Origin 7.0 software. The errors for all the reported thermodynamic parameters were estimated through Monte Carlo simulation with the standard errors of three experiments.