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 fil aments. We have been investigating key interactions of the Ca2+-bindin g protein TnC acid the inhibitory protein TnI. Previously, we used 1-e thyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-lengt h cross-links in the complex of rabbit skeletal muscle TnC and TnI, an d found that the N-terminal, regulatory domain of TnC formed cross-lin ks 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 syntheti c peptide, based on residues 104-115 of TnI, which mimics intact TnI i n its ability to inhibit actomyosin ATPase activity, Prior to cross-li nking, we acetylated the epsilon-amino groups of the nine lysine resid ues of TnC in order to prevent intramolecular cross-linking. Cross-lin ked TnC-peptide products were cleaved with CNBr and several proteinase s. The resulting cross-linked peptides were purified by HPLC and chara cterized by amino-acid sequence analysis. Our results indicate that th e TnI peptide interacted most strongly with two sites in TnC: Glu-60 a nd/or Glu-61 in the N-terminal domain, and acidic residue(s) in segmen t 84-94 of the linker region which connects the N- and C-terminal doma ins of TnC.