AN EXAMINATION OF GLUTAMIC-ACID IN THE -X CHELATING POSITION OF THE HELIX-LOOP-HELIX CALCIUM-BINDING MOTIF

Poor calcium affinity was exhibited in helix-loop-helix calcium bindin g motifs with X-axis acid pairs containing aspartic acid in the -X che lating position. In order to increase interaction of the -X chelating residue with the cation, helix-loop-helix calcium binding motifs were synthesized containing three and four acid residues in chelating posit ions, with a glutamic acid replacing aspartic acid in the -X chelating position. The glutamate-containing motif gave an unexpected g-fold de crease in cation affinity for the three-acid residue loop motif (K-Ca = 524 mu M vs K-Ca = 3140 mu M) and a 46-fold decrease for the four-ac id residue loop motif (K-Ca = 42.1 mu M vs K-Ca 1950 mu M). To improve calcium binding of the glutamate-containing motifs, peptides were syn thesized keeping glutamate in the -X position and inserting serine in the +Z position to provide a hydrogen-bonded system stabilizing the gl utamate interaction with the cation. The serine residue further reduce d calcium affinity in both the three-acid residue loop (K-Ca = 19.6 mM ) and the four-acid residue loop (K-Ca = 2806 mu M) These results indi cate that glutamate and serine residues in the -X and +Z positions, re spectively, can be detrimental to 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 m otifs, but this may be dependent on the environment created by as yet undetermined factors. (C) 1994 Academic Press, Inc.