Covalent binding of peptides to the N-terminal hydrophobic region of cardiac troponin C has limited effects on function.
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.
Department of Biochemistry & Molecular Biology, University of Texas Medical School, Houston 77030, USA.
X
X
Lin
D G
DG
Dotson
J A
JA
Putkey
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D015398:Signal Transduction; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D001710:Biotin; D002118:Calcium