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

Abstract

Two 12-residue peptides were synthesized by the solid-phase method as structural analogs of a Ca 2+-binding loop of rabbit skeletal troponin C. The sequence of the analogs corresponds to the binding loop of the Ca 2+-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 Tb 3+ or large excess of Ca 2+. From the enhancement of Tb 3+ emission association constants in the range (2–3) × 10 5 m −1 and a binding stoichiometry of 1 were determined for Tb 3+ binding to the peptides. Large excess of Ca 2+ displaced Tb 3+ from the Tb 3+-peptide complexes and from these results apparent stability constants of 500–700 m −1 were deduced for Ca 2+ binding. Preliminary proton nuclear magnetic resonance results on one of the peptides indicated that La 3+ 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.