FIGURE 4.

Binding events monitored by ITC. Enthalpies of CcdB binding to GyrA12 (a) and GyrA14 (b) GyrA fragments at various fragment/CcdB molar ratios (r) and temperatures (T) monitored by ITC. The lines represent the best global fits of the models (Equation 6; schematically presented as insets in panels a and b) from which the corresponding thermodynamic parameters were extracted (Table 1 and Fig. 6). c, competition ITC experiment: The GyrA59-CcdB complex was titrated with GyrA14 at T = 25 °C. The line represents the best fit of the displacement model (Equation 6) The obtained best fit thermodynamic parameters ΔG_(T)^o = 1 (± 1) kcal mol⁻¹ and ΔH_(T)^o = 3 (± 2) kcal mol⁻¹ corresponding to the GyrA59-CcdB + GyrA14 ↔ GyrA14-CcdB + GyrA59 process combined with those accompanying CcdB + GyrA14 ↔ GyrA14-CcdB association (Table 1) result in values of ΔG_(T)^o = −13 (±1) kcal mol⁻¹ and ΔH_(T)^o = −10 (±2) kcal mol⁻¹ for CcdB + GyrA59 ↔ CcdB:GyrA59 binding that are in accordance with the corresponding thermodynamic parameters estimated from urea denaturation experiments (Fig. 6). Because of very low heat effects accompanying the titration, we consider this experiment only as an additional support of the physical meaning of the thermodynamic binding parameters presented in Fig. 6.