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 (1) 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 alpha-helical secondary structure and thus isolates the I state. A set of five peptides, alpha alpha-36, beta beta-36, alpha beta-36, gamma beta-36 anti omega omega-36, was generated by shuffling guest hydrophobes at equivalent sites in a symmetric host frame. In one of the peptides, omega omega-36, all the alpha-glutamic residues in the host frame were replaced by gamma-glutamic residues. alpha-36, beta beta-36, alpha beta-36, gamma delta-36 form two-stranded coiled-coils of identical helical content and unfold as a two-state transition during temperature denaturation while the fifth peptide, omega omega-36, is a random coil and cannot be induced in to an oc-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. (C) 2000 Elsevier Science B.V. All rights reserved.