1H-NMR study of Ca(2+)-and Mg(2+)-dependent interaction between troponin C and troponin I inhibitory peptide (96-116).
The Ca(2+)-and Mg(2+)-dependence of the interaction between rabbit skeletal muscle troponin C (TnC) and a 21 residue peptide corresponding to 96-116 of troponin I (denoted as CN4) was examined by means of 1H-NMR spectroscopy. The spectral changes of TnC with 4 mol of Ca2+ (Ca4TnC) and TnC with 4 mol of Mg2+ (Mg4TnC) were observed as a function of CN4 concentration. As CN4 was added to Ca4TnC, resonances of the following residues changed in chemical shift: Tyr10, Phe23, Phe72, Ala106, Gly108, Tyr109, Ile110, His125, Gly144, Ile146, Phe102 or Phe151, and Phe148 located in the N- and C-domains of Ca4TnC. Such CN4-induced change was also observed for resonances of Phe19, 26, and 75 in the N-domain of Ca4TnC by means of NOESY and HOHAHA experiments. The presence of CN4 increased the native-to-unfolded transition temperature of the N-domain of Ca4TnC. On the basis of these results, we conclude that CN4 binds to both the C- and N-domains of Ca4TnC ([CN4]:[TnC] = 1:1) and stabilizes the structure of the N-domain. The CN4-binding constant was estimated to be 1.1 x 10(5) M-1. As CN4 was added to Mg4TnC, chemical shift change was observed for resonances of Phe99, Tyr109, and Ile110 in the C-domain, while no change was observed for resonances arising from the N-domain. The presence of CN4 did not change the thermal stability of the N- and C-domains of Mg4TnC. The CN4-binding constant of Mg4TnC was obtained as 0.9 x 10(4) M-1, which is one-tenth of that of Ca4TnC.(ABSTRACT TRUNCATED AT 250 WORDS)
Tsuda
S
S
High-Resolution Nuclear Magnetic Resonance Laboratory, Faculty of Science, Hokkaido University.
Aimoto
S
S
Hikichi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011522:Protons; D011817:Rabbits; D013696:Temperature; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D002413:Cations, Divalent; D011522:Protons; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D008274:Magnesium; D002118:Calcium