H-NMR study of rabbit skeletal muscle troponin C: Ca(2+)-dependent interaction with mastoparan.
1H-NMR spectroscopy is employed to study the interaction between rabbit skeletal muscle troponin (C (TnC) and wasp venom tetradecapeptide mastoparan. We monitored the spectral change of the following species of TnC as a function of mastoparan concentration: apoTnC, Ca(2+)-saturated TnC (Ca4TnC) and Ca(2+)-half loaded TnC (Ca2TnC). When apo-TnC is titrated with mastoparan, line-broadening is observed for the ring-current shifted resonance of Phe-23, Ile-34, Val-62 and Phe-72 and the downfield-shifted CH alpha-resonances of Asp-33, Thr-69 and Asp-71; these residues are located in the N-domain. When Ca4TnC is titrated with mastoparan, chemical shift change is observed for the ring-current shifted resonances of Phe-99, Ile-110 and Phe-148 and the downfield-shifted CH alpha-resonances of Asn-105, Ala-106, Ile-110 and Ile-146 and aromatic resonance of Tyr-109 and His-125; these residues are located in the C-domain. The resonance of Phe-23, Asp-33, Asp-71, Phe-72, Phe-99, Tyr-109, Ile-146, His-125 and Phe-148 in both N- and C-domains changes when Ca2TnC is titrated with mastoparan. These results suggest that mastoparan binds to the N-domain of apo-TnC, the C-domain of Ca4TnC and the N- and C-domains of Ca2TnC; the hydrophobic cluster in each domain is involved in binding. As mastoparan binds to TnC, the above resonances shift to their normal chemical shift positions. The stability of the cluster and the beta-sheet is reduced by mastoparan-binding. These results suggest that the conformation of the hydrophobic cluster and the neighboring beta-sheet change to a loose form. The stability of the N-domain of Ca2TnC and Ca4TnC increases when these species bind 1 mol of mastoparan at the C-domain. These results suggest a mastoparan-induced interaction between the N- and C-domains of TnC.
Tsuda
S
S
High-Resolution Nuclear Magnetic Resonance Laboratory, Faculty of Science, Hokkaido University, Sapporo, Japan.
Hikichi
K
K
D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D006859:Hydrogen; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009132:Muscles / Q000378:metabolism*; D010455:Peptides; D011487:Protein Conformation; D011817:Rabbits; D013816:Thermodynamics; D014336:Troponin / Q000302:isolation & purification / Q000378:metabolism*; D019209:Troponin C; D014862:Wasp Venoms / Q000378:metabolism*
D001059:Apoproteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014336:Troponin; D019209:Troponin C; D014862:Wasp Venoms; C037500:mastoparan; D006859:Hydrogen; D002118:Calcium