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

University of British Columbia

The acid pair hypothesis predicts the calcium affinity of the helixloop- 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 i-V 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 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. In order to increase interaction of the —X chelating residue with the cation, helix-loop-helix calcium binding motifs were synthesized containing 3 and 4 acid residues in chelating position, with a glutamic acid replacing aspartic acid in the —X chelating position. The glutamate containing motif gave an unexpected 6-fold decrease in cation affinity for the 3 acid residue loop motif and a 46-fold decrease for the 4 acid residue loop motif. To improve calcium binding of the glutamate containing motifs, peptides were synthesized keeping glutamate in the —x position and inserting serine in the +Z position to provide a hydrogen bonded system stabilizing the glutamate interaction with the cation. The serine residue further reduced calcium affinity in both the 3 and 4 acid residue loop. These results indicate that glutamate and serine residues in the —X and +Z position respectively, can have a detrimental effect on calcium binding. However, in natural calcium binding proteins, glutamate in the —X chelating position can confer high affinity for calcium in helix-loop-helix calcium binding motifs, but this may be dependent on the environment created by as yet undetermined factors. 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 residues in X axis acid paired peptides, whereas Z-axis acid paired peptides may 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.

Thesis/Dissertation

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2009-04-14

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http://hdl.handle.net/2429/7070

Pharmaceutical Sciences

University of British Columbia

UBCV

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