Mutational effects on the cooperativity of calcium 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.

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

“…30,64,65 Calmodulin was expressed in E. coli and purified as described. 66 Preparation of Proteins. The freeze-dried CB mutants, scMN+2, scMN-2, and calmodulin were dissolved in the appropriate buffer and centrifuged 7 min at 13 000 g to remove any nondissolved particles.…”

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

“…Calmodulin and Peptides-The CaM expression plasmid pCaM, which contains a synthetic mammalian CaM gene, was a gift from Dr. T. Grundström (University of Umeå, Sweden) and has been described elsewhere (27,30). CaM was purified from Escherichia coli cells containing pCaM by published methods (31)(32)(33).…”

Calmodulin Mediates Brain-derived Neurotrophic Factor Cell Survival Signaling Upstream of Akt Kinase in Embryonic Neocortical Neurons

“…A synthetic gene encoding wild-type bovine calmodulin (38) were all changed to Ala. A 5Ј-flanking BamHI restriction site and a 3Ј-flanking EcoRI restriction site were introduced into the wild-type and mutant calmodulin cDNAs. DNA fragments (BamHI-EcoRI) containing the entire coding region of wild-type or mutant calmodulin were first subcloned into the pGEX-4T plasmid.…”