MODIFIED HELIX-LOOP-HELIX MOTIFS OF CALMODULIN - THE INFLUENCE OF THEEXCHANGE OF HELICAL REGIONS ON CALCIUM-BINDING AFFINITY

The four calcium-binding sites, called the helix-loop-helix, or the EF -hand motifs, of calmodulin differ in their ion-binding affinities; th is 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 hel ical flanks of site 4 (the strongest calcium-binding site of the calmo dulin) 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 hel ical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dy e Stains-all ,5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocyanine bromid e). Binding abilities were seen to increase several-fold when the E an d 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 abso rption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 44 4-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weake n it.