The Changes in Heat Capacity and Entropy of Troponin C Induced by Calcium Binding1

Abstract

We have used the method of enthalpy titration to analyze the structural changes of troponin C caused by calcium binding. Successive additions of calcium to metal-free troponin C in a microcalorimeter cell result in at least three distinguishable transitions. Analysis of the results has shown that troponin C has at least three classes of calcium binding sites; one site of highest affinity (binding constant, 10(8)-10(10) M-1), one site of next highest affinity (binding constant, 10(6)-10(7) M-1) and two low affinity sites (binding constant, 10(5)-10(6) M-1). Titrations of troponin C with calcium at various temperatures have shown that calcium binding causes large changes in the heat capacity of troponin C. Following the method of Sturtevant (1977), the magnitudes of the hydrophobic and intramolecular vibrational contributions to the heat capacity and entropy changes of troponin C on calcium binding have been estimated. In Mg-free solutions, calcium binding to the 1st site of highest affinity gives rise to a strong hydrophobic effect and to a tightening of the molecular structure. In contrast, calcium binding to the 2nd site of next highest affinity gives rise to a strong hydrophobic effect in the reverse direction and to a "softening" of the structure. Calcium binding to the two low affinity sites has a moderately strong hydrophobic effect and also causes a moderate tightening of the structure. These results are in many respects similar to those obtained with proton magnetic resonance spectroscopy by Levine et al. (1977). These studies are mutually complementary. When 1 mM magnesium is present the changes caused by calcium binding to the two high affinity sites are greatly altered, whereas those involving the two low affinity sites are not much affected. The moderate tightening of the structure which is caused by calcium binding to the two low affinity sites, and which is seen both in the absence and presence of magnesium, is most likely to be involved in the regulation of contraction.

Relative stabilities of synthetic peptide homo‐ and heterodimeric troponin‐C domains

“…In the case of the C-terminal domain of TnC, AGO has also been measured calorimetrically for the binding of 2 calcium ions (Potter & Gergely, 1975;Yamada, 1978;Yamada & Kometani, 1982;Imaizumi et al, 1987). Although studies for rabbit TnC are not so clear, a AGO = -85 kJ/mol was obtained for bullfrog TnC (Imaizumi et al, 1987), which is in excellent agreement with the AGO derived from dissociation constants (Table 1).…”

A calorimetric study of Ca2+ binding by the parvalbumin of the toad (Bufo): distinguishable binding sites in the molecule

“…Titrations with Ca2+ (or Mg'+) were performed by the successive addition of a small amount (4.1 pl) of 25 mM CaC12 (or 50 mM MgC12) solutions from a titration apparatus mounted on the outside of the calorimeter block [24,25]. The calorimeter cell contained 5 ml of 0.1-0.18 mM parvalbumin, 100 mM KC1 and 20 mM piperazine-ZV,N'-bis(2-ethanesulfonic acid) (Pipes)-NaOH (pH 7.0).…”

“…Further details of the calorimetric titrations are given in [25-271. To obtain the heat attributable to Ca2+ (or Mg2+) binding, the observed heat must be corrected for the heat caused by the interaction between the Pipes buffer and protons released when Ca2+ (or Mg2+) binds. The latter was determined as in [22,25]; it was less than 0.05 mol/mol binding site. The observed enthalpy changes were not corrected, since the heat actually produced by the pro-78 tons released was negligible, taking the enthalpy change of the protonation of Pipes as -11.46 kJ/mol [28,29].…”

“…Assuming that the toad parvalbumin molecule has two independent Ca2+-binding sites, the observed enthalpy titration curves were analyzed by the least-squares method to estimate the most probable values of the intrinsic binding constant in M-' (ki), the enthalpy change in kJ -(mol site)-' (AH), and the apparent mole number (ni) of ith Ca2+-binding sites [22,25]. The observed heat in kJ -(mol protein)-' (Q) has the opposite sign from the enthalpy change and can be expressed as follows:…”

Heat, Phosphorus NMR and Microcalorimetry in Relation to the Mechanism of Filament Sliding

“…In rabbit skeletal muscle the characteristic phase of positive enthalpy and heat capacity changes is associated with calcium binding to one of the two high-afifinity calcium sites of the C-terminal domain (Yamada and Kometani, 1982). Calorimetric studies have also shown that, in frog skeletal troponin C, one of the N-terminal low-affinity calcium-binding sites is the "active" calcium site (Imaizumi and Tanokura, 1990).…”