PROTEIN DESTABILIZATION BY ELECTROSTATIC REPULSIONS IN THE 2-STRANDEDALPHA-HELICAL COILED-COIL LEUCINE-ZIPPER

The destabilizing effect of electrostatic repulsions on protein stabil ity has been studied by using synthetic two-stranded or-helical coiled -coils as a model system. The native coiled-coil consists of two ident ical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain d isulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil st ructure 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. Four mutant coiled-coils were desig ned to contain one or two Glu substitutions for Gin per polypeptide ch ain. The resulting coiled-coils contained potential i to i' + 5 Glu-Gl u interchain repulsions (denoted as peptide E(2)(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E(2)(20,22)), or no interchain ionic interactions (denoted E(2)(13,22) and E(1)(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfid e bridge. At pH 7, in the presence of urea, the stabilities of E(2)(13 ,22) and E(2)(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E(2)(20,22) coiled-coil do es not occur. In contrast, the mutant E(2)(15,20) is substantially les s stable than E(2)(13,22) or E(2)(15,20) by 0.9 kcal/mol due to the pr esence of two i to i' + 5 interchain Glu-Glu repulsions, which destabi lize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils w ere found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chain s present at low pH are significantly more hydrophobic than Gin side c hains which are in turn more hydrophobic than the ionized Glu side cha ins present at neutral pH. The protonated Glu residues increase the hy drophobicity of the coiled-coil interface leading to higher coiled-coi l stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled-coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ion ic nature and that Glu and Gin in the e and g positions of the heptad repeat have very similar effects on coiled-coil stability in the prese nce of GdnHCl.