Key result
Covalent conjugation of a peptide to Cys-81 in cardiac troponin C greatly decreased myofibril ATPase activity (70-80% inhibition), whereas modification at other sites had lesser or no effect.
Why the study?
Does covalent modification of the N-terminal hydrophobic region of cardiac troponin C affect myofibril ATPase activity?
Does covalent modification of the N-terminal hydrophobic region of cardiac troponin C affect myofibril ATPase activity?
A specific subregion of the N-terminal hydrophobic region in cardiac troponin C is sensitive to disruption, suggesting that Ca2+-sensitizing drugs may bind to this region without interfering with Ca2+ signal transmission.
Cys engineering and adduct attachment in cTnC tests N-domain hydrophobic exposure; leaves open any clinical translation to striated muscle disorders.
Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide specific sites for attachment of blocking groups. A synthetic peptide, Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these Cys residues, and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity (70-80% inhibited) resulted when the peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of inhibition (10-25% inhibited) resulted from covalent modification of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the Ca2+ dependence of ATPase activity was essentially identical to the unmodified protein. Thus, a subregion of the N-terminal hydrophobic region in cTnC is sensitive to disruption, while other regions are less important or can adapt to rather bulky blocking groups. The data suggest that Ca(2+)-sensitizing drugs may bind to the N-terminal hydrophobic region on cTnC but not interfere with transmission of the Ca2+ signal.
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Lin et al. (1996) studied this question. Covalent binding of peptides or biotin to engineered Cys residues in the N-terminal hydrophobic region of cardiac troponin C vs. Unmodified protein was evaluated on Myofibril ATPase activity. Covalent conjugation of a peptide to Cys-81 in cardiac troponin C greatly decreased myofibril ATPase activity (70-80% inhibition), whereas modification at other sites had lesser or no effect.