Determination of and corrections to sequences of turkey and chicken troponins-C. Effects of Thr-130 to Ile mutation on Ca2+ affinity.

Reported differences in the primary structures of chicken muscle troponin C (Wilkinson, J.M. (1976) FEBS Lett. 70, 254-256) and recombinant protein deduced from a chick muscle cDNA (Reinach, F.C. and Karlsson, R. (1988) J. Biol. Chem. 263, 2371-2376) have been reinvestigated. The complete amino acid sequence of turkey muscle troponin C has also been elucidated. Residue 100, originally reported as Asp in the chicken muscle protein, is shown to be Asn in all three structures. The three amino acid sequences are identical except as follows: 1) the blocked NH2-terminal Ala at residue 1 of the chicken protein is replaced by nonblocked Met-Ala in the recombinant protein and by nonblocked Pro in turkey troponin-C; 2) residue 130 is Thr in both avian muscle proteins but Ile in the recombinant protein; 3) Asp-133 in the chicken muscle and recombinant troponins-C is replaced by Glu in the turkey protein; 4) residue 99, originally identified as Glu in the x-ray structure of the turkey protein, is shown to be Ala in all three proteins. Calcium titration of the metal-induced conformational transition of the protein as monitored by far UV CD measurements indicated a significant decrease in Ca2+ affinity of the high-affinity sites in the case of the recombinant protein as compared with the chicken muscle protein. Both pairs of sites showed high cooperativity. That this decreased Ca2+ affinity could be attributed to different amino acid residues at position 130 and not to the differences at the NH2 termini was confirmed by site-specific mutation of Ile-130 to Thr in the recombinant protein. The mutated recombinant protein now titrated identically to the chicken muscle protein. Thr-130, whereas over 21 A from the metal of sites III and IV, is involved in a hydrogen bonding network with structured water and the NH2-terminal region of helix G.