Investigating the role of Ca²⁺-binding site IV in barnacle troponin C

Two genetically engineered, recombinant versions of native barnacle troponin C (TnC) (BTnC₂) were created from the bacterially expressed, recombinant, wild-type BTnC (BTnCWT) to investigate the role of the Ca²⁺- specific sites in force regulation. The mutant BTnC4- contains a single amino acid mutation in site IV which results in the inactivation of site IV Ca²⁺ binding; the mutant BTnCTrunc lacks the last 11 amino acids of the C- terminal, and hence most of site IV. Both mutant proteins, which retain an active site II, bind to native TnC-depleted myofibrillar bundles and restore approximately 40% of the tension-generating capacity, about half that seen with purified native BTnC₁ or BTnC₂. This observation implies that the Mg²⁺-dependent interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca²⁺-binding sites of BTnC₂. Replacement with BTnCTrunc increases the sensitivity of the myofibrillar bundle to changes in ionic strength. Decreasing the ionic strength from 0.15 to 0.075 M increased force by 34%, a value much greater that the 8% increase seen in control bundles or bundles substituted with BTnC4-. These findings implicate TnC in determining this fibre characteristic, although this cannot be simply due to the alteration in the numbers of Ca²⁺ ions bound by the troponin complex since both BTnC4- and BTnCTrunc bind only 1 tool Ca²⁺/mol protein.