The Effects of Interhelical Electrostatic Repulsions between Glutamic Acid Residues in Controlling the Dimerization and Stability of Two-stranded -Helical Coiled-coils

The effects of interhelical electrostatic repulsions in controlling the dimerization and stability of two-stranded α-helical coiled-coils have been studied using de novo designed synthetic coiled-coils. A native coiled-coil was synthesized, which consisted of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain 2-2′ disulfide bridge. This peptide, designed to contain no intrachain or interchain electrostatic interactions, forms a stable coiled-coil structure at 20°C in benign medium (50 mM KCl, 25 mM PO , pH 7) with a [urea] value of 6.1 M. Five mutant coiled-coils were designed in which Gln residues at the e and g positions of the heptad repeat were substituted with Glu systematically from the N terminus toward the C terminus, resulting in each polypeptide chain having 2, 4, 6, 8, or 10 Glu residues. These substituted Glu residues are able to form interchain i to i‘ +5 electrostatic repulsions across the dimer interface. As the number of interchain repulsions increases, a steady loss of helical content is observed by circular dichroism spectroscopy. The effects of the interchain Glu-Glu repulsions on the coiled-coil structure are partly overcome by the presence of an interchain disulfide bridge; the peptide with six Glu substitutions is only 15% helical in the reduced form but 85% helical in the oxidized form. The stabilities of the coiled-coils were determined by urea and guanidine hydrochloride (GdnHCl) denaturation studies at 20°C. The stabilities of the coiled-coils determined by urea denaturation indicate a decrease in stability, which correlates with an increasing number of interchain repulsions ([urea] values ranging from 8.4 to 3.7 M in the presence of 3 M KCl). In contrast, all coiled-coils had similar stabilities when determined by GdnHCl denaturation (approximately 2.9 M). KCl could not effectively screen the effects of interchain repulsions on coiled-coil stability as compared to GdnHCl.