Biochim Biophys Acta Proteins Proteom 2019 Apr 10;1867(4):359-366. Epub 2019 Jan 10.
Department of Chemistry, East Carolina University, 300 Science and Technology, Greenville, NC 27858, United States. Electronic address:
Background: Characterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a "gold-standard" for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems.
Methods: In this study, the thermodynamics of Ca binding to three buffers (Bis-Tris, MES, and MOPS) were obtained from Ca-EDTA titrations using ITC. These data were used to extract buffer-independent parameters for Ca binding to human cardiac troponin C (hcTnC), an EF-hand containing protein required for heart muscle contraction.
Results: The number of protons released upon Ca binding to the C- and N-domain of hcTnC were found to be 1.1 and 1.2, respectively. These values permitted determination of buffer-independent thermodynamic parameters of Ca-hcTnC binding, and the extracted data agreed well among the buffers tested. Both buffer and pH-adjusted parameters were determined for Ca binding to the N-domain of hcTnC and revealed that Ca binding under aqueous conditions and physiological ionic strength is both thermodynamically favorable and driven by entropy.
Conclusions: Taken together, the consistency of these data between buffer systems and the similarity between theoretical and experimental proton release is indicative of the reliability of the method used and the importance of extracting metal-buffer interactions in these studies.
General Significance: The experimental approach described herein is clearly applicable to other metal ions and other EF-hand protein systems.