Amino acid sequences of the two major isoforms of troponin C from crayfish.

The primary structure of the two major isoforms (alpha and gamma) of troponin C (TnC) from crayfish tail muscle has been determined by the application of manual and automated Edman degradation procedures to fragments generated by suitable chemical and proteolytic cleavages. Both amino acid sequences commence with an acetylated methionyl residue and contain 150 amino acid residues, including a single proline residue at position 29 and 2 residues of tyrosine at positions 95 and 102. No cysteine or tryptophan are present. The molecular weights calculated for alpha- and gamma-TnC are 17,157 and 16,974, respectively. The two crayfish proteins are invariable at 129 positions and conserved at 11 others. Pairwise comparisons show that the two sequences are 33-39% identical with those of seven TnCs reported so far and 39% identical with that of bovine brain calmodulin. The N-terminal end of about 10 residues, found in vertebrate TnCs, is absent in crayfish TnCs. In the latter proteins, domains I and III appear as abortive Ca2+-binding sites due to nonconservative amino acid replacements at the key Ca2+-coordinating positions in their loops. The remaining two Ca2+-binding loops (II and IV) show a remarkable similarity with the Ca2+-specific loops (I and II) found in vertebrate TnCs. These findings are consistent with the Ca2+-binding data (Wnuk, W. (1989) J. Biol. Chem. 264, 18240-18246) which indicate the presence of two Ca2+-specific sites in crayfish TnCs. These two sites display the same affinity for Ca2+ (log KCa = 4.3) on gamma-TnC but differ in their affinity (log KCa = 6.0 and 4.1) on alpha-TnC. The only structural difference between the dodecapeptide loops II and IV in both alpha- and gamma-TnC, which correlates with the existence of the high affinity (log KCa = 6.0) Ca2+-specific site on alpha-TnC, is position 11 occupied by a methionyl residue in the loop IV of alpha-TnC as opposed to negatively charged residues found in the other three loops. This suggests that the high affinity Ca2+-specific site on alpha-TnC is located in domain IV. Since the Ca2+-binding studies show that the formation of the complex of crayfish troponin I (TnI) with alpha- and gamma-TnC increases significantly the affinity of only one of their two Ca2+-specific sites and this TnI-sensitive site is not the high affinity Ca2+-specific site on alpha-TnC, we conclude that the binding of Ca2+ to site II controls the Ca2+-dependent interaction between crayfish TnCs and TnI.