title: The effects of interhelical electrostatic repulsions between glutamic acid residues in controlling the dimerization and stability of two-stranded alpha-helical coiled-coils.
abstract: The effects of interhelical electrostatic repulsions in controlling the dimerization and stability of two-stranded alpha-helical coiled-coils have been studied using de novo designed synthetic coiled-coils. A native coiled-coil was snythesized, 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 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 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 degrees 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]1/2 values ranging from 8.4 to 3.7 M in the presence of 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.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: W D
authors.initials: WD
authors.lastname: Kohn
authors.forename: O D
authors.initials: OD
authors.lastname: Monera
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: R S
authors.initials: RS
authors.lastname: Hodges
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013816:Thermodynamics; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
chemical_list: D004220:Disulfides; D006146:Guanidines; D010455:Peptides; D018698:Glutamic Acid; D011189:Potassium Chloride; D014508:Urea; D019791:Guanidine