The relative positions of alanine residues in the hydrophobic core control the formation of two-stranded or four-stranded alpha-helical coiled-coils.

The objective of this study was to investigate the positional effect of hydrophobic interactions in the alpha-helical interface in controlling the formation of two-stranded and four-stranded coiled-coils. Two disulfide-bridged antiparallel coiled-coils were designed which differ only in the position of a single Ala residue in the middle heptad: in peptide 2H the Ala residues are in register (in the same rung), while in peptide 4H they are not. Data from size-exclusion chromatography and sedimentation equilibrium experiments showed that under benign conditions peptides 2H and 4H were two-stranded and four-stranded coiled-coils respectively. These results, in conjunction with molecular modeling studies, suggests that when four Ala residues are in the same plane of a potential four-stranded coiled-coil, the small side chains of Ala would create a large cavity in the hydrophobic interface of the potential four-stranded structure which is destabilizing and favors the two-stranded, disulfide-bridged coiled-coil. In contrast, an alternating Leu-Ala hydrophobic packing in the two planes distributes the potential cavity over a larger region, which may be partially filled by minor adjustments of the neighboring Leu side chains. As a result, there is still sufficient hydrophobic contact to maintain the four stranded structure.