Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal-Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal-protein interactions, including those in metal-Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aβ16 was found to be 1.1 (±0.13) × 10(9) and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aβ28 variants lacking a potential Cu(2+)-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu(2+) binding to Aβ and other intrinsically disordered peptides.

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca(2+) binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin-tropomyosin-actin complex. Herein, we report a calorimetric study of Ca(2+) binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca(2+) binding to both the high-affinity and the low-affinity domain of HcTnC. Ca(2+) binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca(2+) binding to the regulatory domain. This pattern stems from changes that occur at the Ca(2+) site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC(1-89) support this claim. The values of the Gibbs free energy for Ca(2+) binding to the N-domain in the full-length protein and to the isolated domain (HcTnC(1-89)) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (∆C(p)) from a plot of ∆H verses temperature and may provide evidence for positive cooperativity of Ca(2+) binding to the C-domain.