Abstract
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. Ca2+ 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 Ca2+ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca2+ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca2+ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca2+ binding to the regulatory domain. This pattern stems from changes that occur at the Ca2+ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC1–89 support this claim. The values of the Gibbs free energy for Ca2+ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC1–89) 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 Ca2+ binding to the C-domain.
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Acknowledgments
We thank Nick Grossoehme, Dean Wilcox, Andrew Morehead, and Colin Burns for their useful comments and advice. We also give special thanks to Jason King and the Toone laboratory for allowing us to use their calorimeter when ours was being repaired. We thank East Carolina University for the start-up funds for this work.
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Skowronsky, R.A., Schroeter, M., Baxley, T. et al. Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites. J Biol Inorg Chem 18, 49–58 (2013). https://doi.org/10.1007/s00775-012-0948-2
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DOI: https://doi.org/10.1007/s00775-012-0948-2