INTERACTIONS OF STRUCTURAL-C AND REGULATORY-N DOMAINS OF TROPONIN-C WITH REPEATED SEQUENCE MOTIFS IN TROPONIN-I

The actomyosin ATPase inhibitory protein troponin I (TnI) plays a cent ral regulatory role in skeletal and cardiac muscle contraction and rel axation through its calcium-dependent interactions with troponin C (Tn C) and actin. Previously we have demonstrated the utility of F29W and F105W mutants of TnC for measurement of binding affinities of inhibito ry peptide TnI(96-116) to its regulatory N and structural C domains, b oth in isolation and in the intact TnC molecule [Pearlstone, J. R. & S millie, L. B. (1995) Biochemistry 34, 6932-6940], This approach is now extended to fragment TnI(96-148). Curve-fitting analyses of fluoresce nce changes induced in the intact TnC mutants and the isolated N and C domains by increasing [TnI(96-148)] have permitted the assignments of K-D values (designated K-D,K-N and K-D,K-C) to the interaction of TnI (96-148) With the N and C domains, respectively, of intact TnC, Taken together with the previous data for TnI(96-116) binding, it can be con cluded that, within TnI(96-148), residues 96-116 are primarily respons ible for binding to C domain of intact TnC and residues 117-148 to its N domain. Inspection of the available mammalian and avian skeletal mu scle TnI amino acid sequences reveals a previously unrecognized conser ved motif repeated 3-fold, once in the inhibitory peptide region (simi lar to residues 101-114; designated alpha) and twice more in the regio n of residues similar to 121-132 (beta) and similar to 135-146 (gamma) . The number and distribution of these motifs have important structura l implications for the TnI C complex. In the beta motif of cardiac TnI , as compared with skeletal, several changes in charged amino acids ar e suggested as candidates responsible for the greater sensitivity of c ardiac Ca2+-regulated actomyosin to acidic pH as in ischemia.