Figure 4

Binding of Ca²⁺ to NCaBD, -1EF+2EF, and +1EF-2EF as measured by ITC. For each injection, 2-μL of a 3-mM Ca²⁺ solution was mixed in a sample cell containing 37.5 μM NCaBD (upper panel in A) or 75 μM -1EF+2EF (upper panel in B) at 25°C. The heat evolved was recorded during titration. The corrected heats evolved per injection for NCaBD was either fitted to a model with 2 independent binding sites (solid line) or with a model with identical, cooperative binding sites (dashed line) (lower panel in A). The ITC thermogram of -1EF+2EF was fitted with a model to one binding site (lower panel in B). A solution of 30 mM Ca²⁺ was titrated into 75 μM +1EF-2EF and its resulting heat evolved per injection was recorded (upper panel in C). The corrected ITC data was fitted with a single (type) binding site model (lower panel in C). The first point in ITC titration was not included in fitting. Data shown are representative of at least four independent experiments.