Interaction of a troponin I inhibitory peptide with both domains of troponin C.

Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC.