title: Mutational effects on the cooperativity of Ca2+ binding in calmodulin.
abstract: The importance of the aspartate ligand in the +Y Ca2+ coordinating position of two EF-hands of calmodulin has been investigated. Synthetic calmodulin genes were used to produce engineered proteins with the wild-type bovine sequence as well as with aspartate 58 in Ca(2+)-binding site II and/or aspartate 95 in site III changed to asparagine. The macroscopic Ca(2+)-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined from titrations with Ca2+ in the presence of 5,5'-Br2BAPTA. Substitution of aspartate by asparagine in Ca(2+)-binding site II led to a slight increase in the total free energy change on Ca2+ binding, and the cooperativity of Ca2+ binding to the N-terminal sites was substantially increased. The change from aspartate to asparagine in site III decreased the Ca2+ affinity and also appeared to decrease the positive cooperativity between the sites in the C-terminal domain. Thus, identical mutations in sites II and III were found to result in opposite effects. The data imply that involvement of liganding side chains in interactions other than direct calcium attraction and calcium coordination is of considerable importance for the Ca(2+)-binding process, particularly for the cooperativity.
authors.affiliation: Department of Cell and Molecular Biology, University of Umeå, Sweden.
authors.forename: Y
authors.initials: Y
authors.lastname: Waltersson
authors.forename: S
authors.initials: S
authors.lastname: Linse
authors.forename: P
authors.initials: P
authors.lastname: Brodin
authors.forename: T
authors.initials: T
authors.lastname: Grundström
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002118:Calcium