Infrared study of synthetic peptide analogues of the calcium-binding site III of troponin C: The role of helix F of an EF-hand motif

The EF‐hand motif (helix–loop–helix) is a Ca2+‐binding domain that is common among many intracellular Ca2+‐binding proteins. We applied Fourier‐transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17‐residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D‐type amino acid, were investigated to disturb the α‐helical conformation of helix F systematically. These D‐type‐substituted peptides showed no band at about 1555 cm−1 even in the Ca2+‐loaded state although the native peptide (L‐type only) showed a band at about 1555 cm−1 in the Ca2+‐loaded state, which is assigned to the side‐chain COO− group of Glu at the 12th position, serving as the ligand for Ca2+ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca2+ and the side‐chain COO− group of Glu at the 12th position. Implications of the COO− antisymmetric stretch and the amide‐I′ of the synthetic peptide analogues of the Ca2+‐binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.