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

Two genetically engineered, recombinant versions of native barnacle troponi n C (TnC) (BTnC(2)) were created from the bacterially expressed, recombinan t, wild-type BTnC (BTnCWT) to investigate the role of the Ca2+-specific sit es in force regulation. The mutant BTnC4- contains a single amino acid muta tion in sire 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 h ence 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 BTnC(1) or BTnC(2). This observation implies that the Mg2+-dependen t interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca2+-binding sites of BTnC(2). Replacement with BTnCTrunc increa ses 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 bund les substituted with BTnC4-. These findings implicate TnC in determining th is fibre characteristic. although this cannot be simply due to the alterati on in the numbers of Ca2+ ions bound by the troponin complex since both BTn C4- and BTnCTrunc bind only 1 mol a(2+)/mol protein.