The effects on protein stability of negatively charged Glu side chains at different positions along the length of the alpha-helix were investigated in the two-stranded alpha-helical coiled-coil. A native coiled-coil has been designed which consists of two identical 35 residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow the formation of an interchain 2-2' disulphide bridge. This coiled-coil contains no intra- or interchain electrostatic interactions and served as a control for peptides in which Glu was substituted for Gln in the e or g heptad positions. The effect of the substitutions on stability was determined by urea denaturation at 20 degrees C with the degree of unfolding monitored by circular dichroism spectroscopy. A Glu substituted for Gln near the N-terminus in each chain of the coiled-coil stabilizes the coiled-coil at pH 7, consistent with the charge-helix dipole interaction model. This stability increase is modulated by pH change and the addition of salt (KCl or guanidine hydrochloride), confirming the electrostatic nature of the effect. In contrast, Glu substitution in the middle of the helix destabilizes the coiled-coil because of the lower helical propensity and hydrophobicity of Glu compared with Gln at pH7. Taking the intrinsic differences into account, the apparent charge-helix dipole interaction at the N-terminus is approximately 0.35 kcal/mol per Glu substitution. A Glu substitution at the C-terminus destabilizes the coiled-coil more than in the middle owing to the combined effects of intrinsic destabilization and unfavourable charge-helix dipole interaction with the negative pole of the helix dipole. The estimated destabilizing charge-helix dipole interaction of 0.08 kcal/mol is smaller than the stabilizing interaction at the N-terminus. The presence of a 2-2'disulphide bridge appears to have little influence on the magnitude of the charge-helix dipole interactions at either end of the coiled-coil.