Calcium-induced changes in calmodulin structural dynamics and thermodynamics

The thermodynamics of the interaction between Ca2+ and calmodulin (CaM) was examined using isothermal titration calorimetry (ITC). The chemical denaturation of calmodulin was monitored spectroscopically to determine the stability of Ca2+-free (apo) and Ca2+-loaded (holo) CaMs. We explored the conformational and structural dynamics of CaM using amide hydrogen–deuterium (H–D) exchange coupled with Fourier transform infrared (FT-IR) spectroscopy. The results of H–D exchange and FT-IR suggest that CaM activation by Ca2+ binding involves significant conformational changes. The results have also revealed that while the overall conformation of holo-CaM is more stable than that of the apo-CaM, some part of its α-helix structures, most likely the EF-hand domain region, has more solvent exposure, thus, has a faster H–D exchange rate than that of the apo-CaM. The ITC method provides a new strategy for obtaining site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.