The changes in heat capacity and entropy of troponin C induced by calcium binding

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.