Figure 3.

Isothermal titration calorimetry (ITC) binding assays. A and B, ITC measurement of Ca²⁺ binding to CaM_12’-IQ at 27 °C (A) and 37 °C (B). The Ca²⁺ binding isotherms at 27 °C and 37 °C were fit to a two-site and one-site model, respectively. The apparent Ca²⁺ affinity (${\mathrm{K}}_{\mathrm{D}}^{\mathrm{a}\mathrm{p}\mathrm{p}}$) and enthalpy difference (ΔH₁ and ΔH₂) are given in Table 1. The CaM_12’–IQ complex in the sample cell (10 μM at 27 °C or 8.0 μM at 37 °C, 1.5 ml) was titrated with aqueous CaCl₂ (0.23 mM at 27 °C or 0.30 mM at 37 °C) using 35 injections of 10 μl each. C–D, ITC measurement of Ca₂/CaM_12’ binding to IQ at 27 °C (C) and 37 °C (D). The dissociation constant (K_D) and enthalpy difference (ΔH) for Ca₂/CaM_12’ binding to IQ mutants (IQ^WT, IQ^Y1649A, IQ^I1654A, IQ^Y1657D, IQ^F1658D, and IQ^F1658A) are given in Table 3. The binding of Ca₂/CaM_12’ to IQ^K1662E could not be accurately measured by ITC because IQ^K1662E formed aggregated species under the conditions required for ITC. E–H, ITC measurement at 27 °C of Ca₂/CaM_12’ binding to IQ^F1649A (E), IQ^I1654A (F), IQ^Y1657D (G), and IQ^F1658D (H). The IQ peptide concentrations for WT, Y1649A, and I1654A were each 10 μM (27 °C) or 7.0 μM (37 °C) in 1.5 ml in the sample cell for titration with 0.1 mM Ca₂/CaM_12’, and Y1657D and F1658D concentrations were each 50 μM in 1.5 ml for titration with 0.5 mM Ca₂/CaM_12’ using 35 injections of 10 μl each. CaM, calmodulin.