Figure 5.

ITC analysis of Ca²⁺ and Mg²⁺ binding to CML15 (A–C) and CML16 (D–F). Representative isotherms are presented for Ca²⁺ titration into CML samples in either the absence (A,D) or presence (B,E) of 5 mM Mg²⁺. Also presented are Mg²⁺ titrations in a Ca²⁺-free background (C,F) for CML15 and CML16, respectively. Binding was monitored at 30°C in 25 mM HEPES, 100 mM KCl, pH 7.5. The upper panels show raw data from calorimetric titrations, using 5 μL injections, of recombinant CMLs with 600 μM CaCl₂ or 600 μM MgCl₂ as noted above. The lower panels present the corresponding integrated binding isotherm modeled for two Ca²⁺-binding events for CML15 and three Ca²⁺-binding events for CML16. Each isotherm is representative of a minimum of three technical replicates. Protein was used at the following concentrations (A = 26 μM, B = 26 μM, C = 31 μM, D = 23 μM, E = 19 μM, F = 23 μM).