Thermodynamics of Calcium Binding to Zebrafish Troponin C (TnC) Visualized through Isothermal Titration Calorimetry (ITC) and Molecular Dynamics (MD)

Abstract

Interaction of calcium Ca2+ and troponin C (TnC) is a fundamental process governing regulation of cardiac contractility. Increased affinity of cTnC's regulatory site II for Ca2+ elevates sensitivity, leading to greater force production capabilities. Maintenance of cardiac function in the face of varying temperature is an important property of the heart and sequence based changes in TnC may alter the ability to modulate force with changing temperature. Two distinct genes express TnC in the teleost heart: cardiac (cTnC) and fish-specific slow skeletal (ssTnC). 18 of 161 amino acids differ between zebrafish paralogs.qPCR experiments in temperature-acclimated zebrafish at 28°C showed ssTnC mRNA levels to be two-fold greater than cTnC mRNA levels while at 18°C, cTnC expression was 1.5-fold higher in the atrium.Isothermal Titration Calorimetry (ITC) was used to study the heat change upon titration of Ca2+ with site II in the N-terminal domain of zebrafish TnC paralogs. The interaction of Ca2+ with TnC was an endothermic, entropically driven reaction. Our work suggests that ssTnC may have a higher Ca2+ affinity (Kd ∼11 and 14 vs. 13 and 17 μM in cTnC at 28 and 18 °C, respectively) at both temperatures indicating a more favorable Gibbs free energy of interaction.The effect of sequence variations between paralogs have been modeled through equilibrium Molecular Dynamics (MD) simulations. The resultant structures were similar, however a measurable difference was seen in the free energy of Ca2+ binding. ΔG of binding for the ssTnC is lower at 18°C and higher at 28°C than the cardiac paralog. Taken together, these results suggest that the increase in expression of the ssTnC at higher temperatures compensates for the decrease in the ability of the cTnC to bind calcium.