Insights into the Intramolecular Coupling between the N- and C-Domains of Troponin C Derived from High-Pressure, Fluorescence, Nuclear Magnetic Resonance, and Small-Angle X-ray Scattering Studies

Troponin C (TnC), the Ca2+-binding component of the troponin complex of vertebrate skeletal muscle, consists of two structurally homologous domains, the N- and C-domains; these domains are connected by an exposed alpha-helix. Mutants of full-length TnC and of its isolated domains have been constructed using site-directed mutagenesis to replace different Phe residues with Trp. Previous studies utilizing these mutants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and that the N-domain has no effect on the stability of the C-domain [Rocha, C. B., Suarez, M. C., Yu, A., Ballard, L., Sorenson, M. M., Foguel, D., and Silva, J. L. (2008) Biochemistry 47, 5047-5058]. Here, we analyzed the stability of full-length F29W TnC using structural approaches under conditions of added urea and hydrostatic pressure denaturation; F29W TnC is a fluorescent mutant, in which Phe 29, located in the N-domain, was replaced with Trp. From these experiments, we calculated the thermodynamic parameters (AV and G a,) that govern the folding of the intact F29W TnC in the absence or presence of Ca2+. We found that the C-domain has only a small effect on the structure of the N-domain in the absence of Ca2+. However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca2+-bound state (i.e., when Ca2+ was also bound to sites III and IV of the C-domain). An accompanying decrease in the thermodynamic stability of the N-domain generated a reduction in Delta Delta G degrees(atm) in absolute terms, and Ca2+ binding affects the Ca2+ affinity of the N-domain in full-length TnC. Cross-talk between the C- and N-domains may be mediated by the central helix, which has a smaller volume and likely greater rigidity and stability following binding of Ca2+ to the EF-hand sites, as determined by our construction of low-resolution three-dimensional models from the small-angle X-ray scattering data.