The aim of the present investigation is to determine the effect of alpha-helical propensity and sidechain hydrophobicity on the stability of amphipathic alpha-helices. Accordingly, a series of 18-residue amphipathic alpha-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic alpha-helix. In these experiments, all three parameters (sidechain hydrophobicity, alpha-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in alpha-helical propensity between the most (Ala) and the least (Gly) alpha-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3 degrees C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and alpha-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic alpha-helix. More importantly, the combined effects of alpha-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with alpha-helix stability. These results suggest that both alpha-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of alpha-helical proteins with the desired stability.

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCI) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.

The role of interchain electrostatic interactions in orientating alpha-helical chains to form two-stranded parallel and antiparallel coiled-coils has been investigated. Four disulfide-bridged coiled-coils were designed: parallel coiled-coils with interchain electrostatic attractions (P/A) and repulsions (P/R) and antiparallel coiled-coils with interchain electrostatic attractions (AP/A) and repulsions (AP/R). These coiled-coils were made by air oxidation of two 35-residue peptides with the appropriate heptad repeat (LaEbAcLdEeGfKg or LaAbEcLdKeGfEg) to give the desired interchain electrostatic interactions, and the appropriate position of the cysteine residue (C2 or C33) to give the desired chain orientation. The coiled-coils were characterized by circular dichroism spectroscopy, and their stabilities were assessed by guanidine hydrochloride and urea denaturations. The results indicated that the favored chain orientation, that is, the major disulfide-bridged product formed under benign conditions, was the one that provides interchain electrostatic attractions between oppositely-charged amino acid residues in the e-g' and g-e' positions of the parallel coiled-coil and the g-g' and e-e' positions in the antiparallel coiled-coil. When the electrostatic interactions were similar, the antiparallel coiled-coils were more stable than the parallel coiled-coils. However, the overall stability of the coiled-coils was either increased by interchain electrostatic attractions or decreased by interchain electrostatic repulsions, as determined by urea denaturation. Thus, the order of overall stability of these coiled-coils was AP/A > P/A > AP/R > P/R. This study demonstrates the importance of interchain electrostatic interactions in determining the parallel or antiparallel orientation of alpha-helical chains in two-stranded coiled-coils.

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The effects of interhelical electrostatic repulsions in controlling the dimerization and stability of twostranded ␣-helical coiled-coils have been studied using de novo designed synthetic coiled-coils. A native coiledcoil 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 coiledcoil structure at 20°C in benign medium (50 mM KCl, 25 mM PO 4 , 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°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 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.

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The orientation of alpha-helical chains in two-stranded coiled-coils has been shown to be determined by the presence of favorable interchain electrostatic interactions. In this study, we used de novo designed 35-residue peptides to show that when interchain electrostatic interactions are not a factor in coiled-coil formation, the relative positions of Ala residues in the middle heptad can control the parallel or antiparallel orientation of alpha-helical chains in coiled-coils. The peptides formed four-stranded coiled-coils where the helices are either all-parallel or all-antiparallel with respect to their nearest neighbor. The common structural element in these four-stranded coiled-coils is an alternating pair of Ala and Leu residues (Ala-Leu-Ala-Leu) in each of the two planes in the middle heptad. These results indicate that both the relative positions of the Ala residues in the hydrophobic core and the interchain electrostatic interactions between charged residues in the e and g positions should be considered in designing coiled-coils with the desired number of strands in the multiple-stranded assembly. These design elements are also important in orienting functional groups or domains attached to the terminals ends of a coiled-coil carrier.

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