title: Free-energy linkage between folding and calcium binding in EF-hand proteins.
abstract: Troponin is the singular Ca(2+)-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca(2+)-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca(2+) was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (DeltaV and DeltaG(atm)). In this study, we propose that part of the affinity for Ca(2+) is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca(2+) binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca(2+) in EF-hand proteins, which is crucial to function.
authors.affiliation: UEZO-Centro Universitário Estadual da Zona Oeste, Rio de Janeiro, Brazil.
authors.forename: Marisa C
authors.initials: MC
authors.lastname: Suarez
authors.forename: Cristiane B
authors.initials: CB
authors.lastname: Rocha
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
authors.forename: Jerson L
authors.initials: JL
authors.lastname: Silva
authors.forename: Debora
authors.initials: D
authors.lastname: Foguel
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D003198:Computer Simulation; D020832:EF Hand Motifs; D004735:Energy Transfer; D019277:Entropy; D007536:Isomerism; D008956:Models, Chemical; D011485:Protein Binding; D017510:Protein Folding; D019209:Troponin C / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D019209:Troponin C; D002118:Calcium