The functional role of the domains of troponin-C investigated with thrombin fragments of troponin-C reconstituted into skinned muscle fibers

Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca²⁺-binding site(s): TH₁ (residues 1-120) sites I-III; TH₂ (121-159) site IV; TH₃ (1- 100) sites I and II; and TH₄ (101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH₁ was the most effective in the force restoration (65%) whereas the smaller fragments developed about 50% (TH₃ and TH₄) or 20% (TH₂) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH₂, TH₃, and TH₄ compared to RSTnC or TH₁ were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH₁ was able to interact with the fibers in a Ca²⁺-independent (Mg²⁺-dependent) manner, indicating that at a minimum, Ca²⁺-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca²⁺. TH₂ and TH₄ appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH₂ and TH₄ was abolished when Ca²⁺ was removed. TH₂ and TH₄ binding to the second site required Ca²⁺ initially but was maintained in the presence of Mg²⁺. This interaction of TH₂ and TH₄ partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III or IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca²⁺-Mg²⁺ sites (III and IV) of TnC.