A structure/activity study of calcium affinity and selectivity using a synthetic peptide model of the helix-loop-helix calcium-binding motif.

The acid pair hypothesis predicts the calcium affinity of the helix-loop-helix calcium-binding motif based on the number and location of acidic amino acid residues in chelating positions of the calcium-binding loop region. This study investigates the effects of the number and position of acidic residues in the loop region on calcium affinity and selectivity using 33-residue synthetic models of single helix-loop-helix calcium-binding motifs. Increasing the number of acidic residues in the octahedrally arranged chelating positions of the loop region from 3 to 4 by replacing an asparagine in the +y position with an aspartic acid increases the calcium affinity of the models between 2- and 38-fold. Differences in affinities are more pronounced in the models containing an x axis acid pair. The calcium affinities of peptide models containing 3 or 4 acidic residues in chelating positions of the loop region and an x axis acid pair are reduced when the residue in the +z position is changed from asparagine to serine. A similar reduction in calcium affinity occurs in the z axis acid paired peptides when the -x chelating residue is changed from serine to asparagine. Models with 3 acidic residues in chelating positions containing a z axis acid pair have greater calcium affinity than comparable peptide models with an x axis acid pair. The presence of x or z axis acid pairs in comparable peptides containing 4 acidic residues in chelating positions does not greatly alter calcium affinity. Calcium selectivity resides in x axis acid paired peptides, whereas z axis acid paired peptides exhibit both magnesium- and calcium-induced structural changes. This ion selectivity may be explained by postulating that the z axis residue side chains produce the initial, rate-limiting interactions with the cation, causing hydration shell destabilization and initiating the subsequent ligand interactions.