AbstractWe have generated mutants of Drosophila calmodulin in which pairs of calcium‐binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the —Z position glutamate residue with glutamine. Mutants inactivated in both N‐terminal sites (B12Q) or both C‐terminal sites (B34Q), and two mutants with one N‐ and one C‐terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV‐difference spectroscopy and near‐UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr‐138) of the protein. These studies uncovered four situations in which Tyr‐138 in the C‐terminal lobe responds to a change in the calcium‐binding properties of the N‐terminal lobe. Further, they suggest that N‐terminal calcium‐binding events contribute strongly to the aberrant behavior of Tyr‐138 seen in mutants with a single functional C‐terminal calcium‐binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr‐138 produced by mutation of site 3 toward the wild‐type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK‐MLCK) established that all of the multiple binding site mutants are poor activators of SK‐MLCK. Thus, globally, the calcium‐induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr‐138 within the crystal structure of calmodulin suggests that effects of the N‐terminal lobe on this residue may be mediated via changes to the central linker region of the protein.