title: Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament.
abstract: The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Av Bauhinia 400 CCS bloco E sala 42, 21941-590 Rio de Janeiro, RJ, Brazil.
authors.forename: Carolina A C A
authors.initials: CA
authors.lastname: Braga
authors.forename: José Renato
authors.initials: JR
authors.lastname: Pinto
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Valente
authors.forename: Jerson L
authors.initials: JL
authors.lastname: Silva
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
authors.forename: Débora
authors.initials: D
authors.lastname: Foguel
authors.forename: Marisa C
authors.initials: MC
authors.lastname: Suarez
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D057927:Hydrophobic and Hydrophilic Interactions; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D019209:Troponin C; D008274:Magnesium; D002118:Calcium