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

Abstract

Troponin C (TnC), the Ca(2+)-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 α-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 (ΔV and ΔG°(atm)) that govern the folding of the intact F29W TnC in the absence or presence of Ca(2+). We found that the C-domain has only a small effect on the structure of the N-domain in the absence of Ca(2+). However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca(2+)-bound state (i.e., when Ca(2+) 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 ΔΔG°(atm) in absolute terms, and Ca(2+) binding affects the Ca(2+) 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 Ca(2+) to the EF-hand sites, as determined by our construction of low-resolution three-dimensional models from the small-angle X-ray scattering data.

Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C

“…Multiple studies of different TnC species and domain fragments provide evidence for interdomain communication 4, 6, 15, 39 . How does the communication between N- and C-domains occur?…”

“…Since Ca 2+ binding to cTnC leads to global structural changes 13, 14 , we postulated that stability and folding might be disrupted, and we sought to correlate the stability of the HcTnC D145E with its physiological function in skinned fibres. For skeletal TnC, several reports relate protein folding and function 15, 16 , whereas for cardiac TnC, folding and stability 6 have attracted less attention than quaternary structure 17 . Here we used nuclear magnetic resonance (NMR) of the recombinant HCM protein to obtain the backbone assignment, and we evaluated its stability and structural features by NMR and circular dichroism (CD) at different temperatures.…”

A hypothesis to reconcile the physical and chemical unfolding of proteins

“…In the case of apomyoglobin, although separate elegant works on pressure denaturation (56) and urea denaturation (57) exist, NMR data have been extensively obtained at 8 M urea and not at intermediate urea concentrations. Considerable fluorescence and NMR data are available for the urea and pressure denaturation of the N and C domains of troponin C (24,(58)(59)(60). Clearly, MpNep2 is highly sensitive to urea and pressure.…”

“…To better understand local changes, high-pressure NMR (HP-NMR) is adopted as the most informative approach (18,19). SAXS is another useful tool for assessing changes in protein folding and the size and shape of macromolecules in solution (24)(25)(26).…”

Allosteric Transmission Along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with only One Ca 2+ ion

“…an increase in protein stability leads to a decrease in Ca 2ϩ affinity (22,23) and vice versa. Intramolecular coupling between the N-and C-domains has been observed for skeletal TnC (24), and several lines of evidence support this cross-talk mechanism (25)(26)(27)(28), which is probably aided by the bending of the DE-linker helix (29,30). Bending at the DE linker is even more pronounced for the cardiac protein (31,32), and we see the consequences of this N-/C-domain cross-talk in the form of an allosteric event in the N-domain, mitigating in part the potentially catastrophic effects of an HCM-related mutation in the C-domain.…”