MUTATIONAL EFFECTS ON THE COOPERATIVITY OF CA2+ BINDING IN CALMODULIN

The importance of the aspartate ligand in the +Y Ca2+ coordinating pos ition of two EF-hands of calmodulin has been investigated. Synthetic c almodulin genes were used to produce engineered proteins with the wild -type bovine sequence as well as with aspartate 58 in Ca2+-binding sit e II and/or aspartate 95 in site III changed to asparagine. The macros copic Ca2+-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined fro m titrations with Ca2+ in the presence of 5,5'-Br2BAPTA. Substitution of aspartate by asparagine in Ca2+-binding site II led to a slight inc rease in the total free energy change on Ca2+ binding, and the coopera tivity of Ca2+ binding to the N-terminal sites was substantially incre ased. The change from aspartate to asparagine in site III decreased th e Ca2+ affinity and also appeared to decrease the positive cooperativi ty between the sites in the C-terminal domain. Thus, identical mutatio ns in sites II and III were found to result in opposite effects. The d ata imply that involvement of liganding side chains in interactions ot her than direct calcium attraction and calcium coordination is of cons iderable importance for the Ca2+-binding process, particularly for the cooperativity.