Article ID Journal Published Year Pages File Type
1984829 The International Journal of Biochemistry & Cell Biology 2006 13 Pages PDF
Abstract

The C-domain of troponin C, the Ca2+-binding subunit of the troponin complex, has two high-affinity sites for Ca2+ that also bind Mg2+ (Ca2+/Mg2+ sites), whereas the N-domain has two low-affinity sites for Ca2+. Two more sites that bind Mg2+ with very low affinity (Ka < 103 M−1) have been detected by several laboratories but have not been localized or studied in any detail.Here we investigated the effects of Ca2+ and Mg2+ 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 Ca2+ or Mg2+ (Ca2+/Mg2+ 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 Ca2+ + Mg2+ were both present or when Ca2+ was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca2+] in two well-defined steps: one at low [Ca2+], consistent with the Ca2+/Mg2+ sites (Ka ∼ 1.5 × 106 M−1), and one of much lower affinity (Ka ∼ 52.3 M−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 Ca2+ (0.6 mM) to occupy the Ca2+/Mg2+ sites, a large increase in bis-ANS binding also occurred as Mg2+ occupied a class of low-affinity sites (Ka ∼ 59 M−1). In skinned fibers, a high concentration of Mg2+ (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.

Related Topics
Life Sciences Biochemistry, Genetics and Molecular Biology Biochemistry
Authors
, , , , , , ,