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) X 10(5) M-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.
D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D009682:Magnetic Resonance Spectroscopy; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D000596:Amino Acids; D009124:Muscle Proteins; D010455:Peptides; D014336:Troponin; D019209:Troponin C; D002118:Calcium