Calcium-induced protein folding. Structure-affinity relationships in synthetic analogs of the helix-loop-helix calcium binding unit.

Three peptide analogs of the helix-loop-helix Ca2+ binding unit, 21-, 26-, and 34-residues in length, similar in sequence to rabbit skeletal troponin C site III have been prepared by the solid-phase method. The CD spectra of the 21-residue fragment indicated very little secondary structure in aqueous medium in the absence of Ca2+. Addition of Ca2+ increased the secondary structure of the peptide but the KCa was very weak, 3.1 x 10(2) M-1. The same peptide in hydrophobic medium in the absence of Ca2+ had considerable secondary structure and the KCa value increased considerably, 3.5 x 10(5) M-1. The 26-residue peptide, containing 5 more residues on the NH2 terminus of the 21-residue peptide, showed slightly more secondary structure in aqueous medium in the absence of Ca2+. Addition of Ca2+ to this peptide raised the amount of secondary structure in the metal ion-peptide complex and resulted in a higher KCa value, 3.8 x 10(4) M-1. By assuming that the COOH-terminal region of the 26-residue peptide-metal ion complex assumes a structure similar to that of the 21-residue peptide-metal ion complex, one is able to assign the increase in structure to the NH2-terminal side of the Ca2+-binding loop. Hydrophobic medium further increased the secondary structure of this peptide and also increased the KCa value to 4.5 x 10(5) M-1, a value similar to that obtained for the 21-residue peptide. The 34-residue peptide contained a further 8 amino acid residues on the NH2 terminus of the 26-residue peptide. This peptide had considerable secondary structure in aqueous medium which increased in the presence of Ca2+. The peptide has a reasonable affinity for Ca2+ in aqueous medium, KCa = 2.6 x 10(5) M-1. Again, a hydrophobic medium increased both the amount of secondary structure and the Ca2+ affinity constant, KCa = 9.2 x 10(5) M-1. A model of Ca2+-induced folding of the three peptides under different conditions is described and results obtained from this model are used to describe Ca2+ binding to the four Ca2+ binding units in rabbit skeletal troponin C.