A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification, and calcium binding.
The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure of the calcium binding sites in the naturally occurring calcium binding protein to the calcium affinity of these sites, the synthetic analogue is examined for calcium binding by circular dichroism spectroscopy. The calcium binding characteristics are compared to those of a synthetic analogue of the homologous calcium binding site III in rabbit skeletal troponin C. The Kd of the calmodulin site III fragment for Ca2+ is determined as 878 microM whereas the Kd of the troponin C fragment is 30 times smaller at 28 microM. Structural changes induced in the peptides by Ca2+ and trifluoroethanol are similar. This study supports our contention that the single synthetic calcium binding site is a reasonable model for the study of the structure-activity relationships of the calcium binding sites in calcium-regulated proteins such as calmodulin and troponin C.
Faculty of Pharmacy, University of Manitoba, Winnipeg, Canada.
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D002147:Calmodulin; D010446:Peptide Fragments; C054438:calmodulin (8-13); D002118:Calcium