Synthetic peptide analogs of skeletal troponin C: Fluorescence studies of analogs of the low-affinity calcium-binding site II

Two 12-residue peptides were synthesized by the solid-phase method as structural analogs of a Ca²⁺-binding loop of rabbit skeletal troponin C. The sequence of the analogs corresponds to the binding loop of the Ca²⁺-specific low affinity binding site II (residues 63-74) but with two amino acid substitutions. In one analog, Phe-72 was replaced by tyrosine. In the other Gly-66 was substituted by serine and Phe-72 by tyrosine. The intrinsic fluorescence of the peptides was enhanced upon addition of Tb³⁺ or large excess of Ca²⁺. From the enhancement of Tb³⁺ emission association constants in the range (2-3) × 10⁵m^-1 and a binding stoichiometry of 1 were determined for Tb³⁺ binding to the peptides. Large excess of Ca²⁺ displaced Tb³⁺ from the Tb³⁺-peptide complexes and from these results apparent stability constants of 500-700 m^-1 were deduced for Ca²⁺ binding. Preliminary proton nuclear magnetic resonance results on one of the peptides indicated that La³⁺ induced considerable perturbation of the amide proton resonances of several residues, including the aspartate at position 3, the tyrosine at position 10, and the two glutamates at the C-terminus. The results suggest involvement of these residues in cation coordination.