Conformational Dynamics of the Regulatory Domain of Troponin C

Abstract

In striated muscles contraction is regulated by the F-actin filament associated troponin-tropomyosin complex. Ca2+-binding to the N-terminal domain of troponin C (TnC) causes opening of that domain and exposure of the binding site for troponin I. To estimate the rate of this transition we have used a mutant of sTnC (TnC48/82) having Cys residues substituted for Gln48 in the B/C linker and Gln82 in helix D. These Cys residues are 4.4 A and 14.3 A apart (Cβ distance) in the closed and open domain conformation, respectively. Thus, they can readily form an intramolecular disulfide bond in the apo, but not in the Ca2+-bound state. We have evaluated the rates and the products of sTnC48/82 reaction with 5,5’-dithio-2-nitrobenzoic acid (DTNB) in a broad concentration range (0.05 - 50.0 mM). The reaction proceeds in two steps. First the thionitrobenzoic moiety is attached to one of the SH groups in a bimolecular disulfide exchange reaction. The second step is either an intramolecular disulfide exchange leading to crosslinking or blocking the remaining SH group with another bimolecular collision with DTNB. The ratio of the products is determined by urea-PAGE. We have found that in the absence of Ca2+ the reaction is slow and leads to 100% disulfide crosslinked product even at the highest DTNB concentrations. Addition of 1 mM CaCl2 causes a large (∼150 fold) increase in the rate of reaction, and surprisingly, at lower DTNB concentrations the protein is still efficiently crosslinked, despite its open conformation. Only at [DTNB]> 20 mM does rate of blocking the remaining Cys residue exceed that of intramolecular crosslinking. These results suggest that the regulatory domain of sTnC is very dynamic, a property that contributes to the rapid on-off switching of muscle contractile activity.