De novo design of a model peptide sequence to examine the effects of single amino acid substitutions in the hydrophobic core on both stability and oligomerization state of coiled-coils

Abstract

A model peptide sequence was de novo designed to investigate the hydrophobicity, stability and oligomerization state resulting from single amino acid substitutions in the hydrophobic core a position of the central heptad of a five heptad coiled-coil. This involved selecting a hydrophobic core consisting of Val and Leu at a and d positions, respectively, known to form both two- and three-stranded coiled-coils. In addition, the sequence provided the correct overall coiled-coil stability and maximized the hydrophobicity surrounding the substitution site by having Leu in the hydrophobic core above and below the site of substitution. To control oligomerization state we exploited differential placement of an interhelical disulfide-bridge, which was placed in the coiled-coil hydrophobic core at the C-terminal d position, or alternatively outside of the core at the N-terminal via a Cys-Gly-Gly linker. We found that the Cys-Gly-Gly linker allowed assessment of both relative stability and oligomerization state after extensive biophysical characterization of the models by circular dichroism, sedimentation equilibrium, sedimentation velocity and finally high-performance size-exclusion chromatography (HPSEC) under both benign and denaturing conditions. The Cys-Gly-Gly linker was found to be unique in allowing the inherent two- or three-stranded oligomerization state to be observed in benign medium, while also allowing the stability to be determined by concentration independent chemical denaturation of a two-stranded coiled-coil. This entails a two-state transition from a folded disulfide-bridged two-stranded coiled-coil (monomeric state) to the unfolded monomer, even for analogs where the coiled-coil is a trimer of disulfide-bridged peptides in benign medium. We also developed novel HPSEC methodology for monitoring the chemical denaturation of a folded monomeric protein in fast exchange with the corresponding unfolded protein, which elutes as a single peak throughout the denaturation process.