Trapping the monomeric α-helical state during unfolding of coiled-coils by reversed-phase liquid chromatography

Reversed-phase liquid chromatography (RPLC) offers a unique opportunity to monitor the transition from the native state (N) to the structural intermediate state (I) for proteins whose secondary structure is comprised entirely of amphipathic helices, such as coiled-coils. During RPLC, the hydrophobicity of the stationary phase and mobile phase results in the unfolding of the tertiary/quaternary structure of coiled-coils but retains α-helical secondary structure and thus isolates the I state. A set of five peptides, αα-36, ββ-36, αβ-36, γδ-36 and ωω-36, was generated by shuffling guest hydrophobes at equivalent sites in a symmetric host frame. In one of the peptides, ωω-36, all the α-glutamic residues in the host frame were replaced by γ-glutamic residues. αα-36, ββ-36, αβ-36, γδ-36 form two-stranded coiled-coils of identical helical content and unfold as a two-state transition during temperature denaturation while the fifth peptide, ωω-36, is a random coil and cannot be induced in to an α-helical structure even in the presence of a helix inducing solvent, 50% trifluoroethanol. By comparing the stability order of the four coiled-coils in the N→I transition (measured by RPLC studies) with that in the N→D (denatured state) transition (measured by calorimetry), it is concluded that there is a direct correlation between the relative stabilities of these peptides in these two unfolding transitions. This result supports a hierarchical folding mechanism for coiled-coils.