title: 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.
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.
authors.forename: Guilherme A P
authors.initials: GA
authors.lastname: de Oliveira
authors.forename: Cristiane B
authors.initials: CB
authors.lastname: Rocha
authors.forename: Mayra de A
authors.initials: Mde A
authors.lastname: Marques
authors.forename: Yraima
authors.initials: Y
authors.lastname: Cordeiro
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
authors.forename: Débora
authors.initials: D
authors.lastname: Foguel
authors.forename: Jerson L
authors.initials: JL
authors.lastname: Silva
authors.forename: Marisa C
authors.initials: MC
authors.lastname: Suarez
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011312:Pressure; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D019209:Troponin C; D014508:Urea; D002118:Calcium