H-1-NMR RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE, AND GLOBAL FOLD OF THE TR1C FRAGMENT OF TURKEY SKELETAL TROPONIN-C IN THE CALCIUM-FREE STATE

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the H-1-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D H-1-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard ''EF-hand'' motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M. N. G. (1988) J. Mol. Biol. 203, 761-779].