Fig. 4. The activation of EF and CyaA-N by wild-type CaM and two series of CaM mutants. EF (1 nM) and CyaA-N (0.7 nM) were used for an adenylyl cyclase activity assay in the presence of 0.1 µM free Ca²⁺ with CaM mutants CaM 41/75 and 85/112. Each mutant has two cysteine mutations to lock either the N- or the C-terminal domain of CaM in the closed conformation (A and B). The same concentrations of EF and CyaA-N were used for an adenylyl cyclase activity assay in the presence of 1 µM free Ca²⁺ with CaM mutants B1Q, B2Q, B3Q and B4Q. Each mutant has a mutation inactivating one of four calcium-binding sites (C and D). Means ± SE are representative of at least two experiments.