Figure 4.

Mg²⁺ binding to the CaMC DD mutant. (A) Titration with Mg²⁺. Superimposition of the ¹H-¹⁵N HSQC spectra of the CaMC DD mutant with Mg²⁺ concentrations ranging from 0 to 100 mM. Each spectrum was acquired at 50°C. (B) Titration curve for Mg²⁺ binding to the CaMC DD mutant. Amide proton chemical shifts assigned to G96 (EF-hand III, circles) and G132 (EF-hand IV, squares), plotted as a function of the Mg²⁺ concentration. (C) Calculated populations of the different states of the protein, as a function of the Mg²⁺ concentration. The populations were calculated for K_III = 29.4 mM, K_IV = 2.67 mM, and 1 mM protein. (D) Backbone amide chemical shift changes induced by Mg²⁺ binding. Black bars represent the chemical shift changes between 0 and 10 mM Mg²⁺, and magenta bars indicate the chemical shift changes between 10 and 100 mM Mg²⁺. The values were calculated with the following equation: . The signals of E83, I85, N111, E114, V142, and Q143 were overlapped. The chemical shift changes of the D95, N97, G98, I100, V108, R126, A128, D131, D133, G134, and V136 residues were too broad, and are not included in the plot. The binding sequences of the EF-hands are indicated with a line. The helices are indicated by boxes with their names.