The four calcium-binding sites, called the helix-loop-helix, or the EF-hand motifs, of calmodulin differ in their ion-binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium-binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium-binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocya-nine bromide). Binding abilities were seen to increase several-fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111-414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it
