Effects of side chains in helix nucleation differ from helix propagation

Abstract

Helix-coil transition theory connects observable properties of the α-helix to an ensemble of microstates and provides a foundation for analyzing secondary structure formation in proteins. Classical models account for cooperative helix formation in terms of an energetically demanding nucleation event (described by the σ constant) followed by a more facile propagation reaction, with corresponding s constants that are sequence dependent. Extensive studies of folding and unfolding in model peptides have led to the determination of the propagation constants for amino acids. However, the role of individual side chains in helix nucleation has not been separately accessible, so the σ constant is treated as independent of sequence. We describe here a synthetic model that allows the assessment of the role of individual amino acids in helix nucleation. Studies with this model lead to the surprising conclusion that widely accepted scales of helical propensity are not predictive of helix nucleation. Residues known to be helix stabilizers or breakers in propagation have only a tenuous relationship to residues that favor or disfavor helix nucleation.

Keywords: helix propensity; synthetic helices.

Fig. 1.

Preorganization of three residues into an α-turn conformation is the energy-demanding step in helix formation. (A) Models of the helix–coil transition consider helix formation to proceed in two steps consisting of nucleation and propagation steps. (B) In hydrogen bond surrogate (HBS) α-helices, the nucleus is organized by replacement of a main chain i to i + 4 hydrogen bond with a covalent bond. The dsHBS helices feature a reversible disulfide linkage. (C) Possible intermediates for the conversion of bisthiol I to dsHBS IV include bisthiol II in α-turn conformation and disulfide III as the constrained helix nucleus. Rates of conversion of bisthiol to disulfide were measured as a function of variable residue Λ.

Fig. 2.

Conformational analysis and synthesis of unconstrained peptides. (A) CD spectroscopy suggests that the disulfide-linked (ds) HBS peptide is highly helical compared with the parent bisthiol (bt). The CD studies were performed with 50 μM peptides in 1 mM PBS. (B) NMR-derived structures of ds-2A. Views of 20 lowest energy structures are shown with carbon, nitrogen, and oxygen atoms in gray, blue, and red, respectively. The disulfide linkage is shown in yellow color. (C) Schematic for the oxidation reaction. The conversion of bt-2Λ → ds-2Λ was affected under mild oxidative conditions; the unreacted bisthiol was quenched with maleimide 3.

Fig. 3.

Analysis of bisthiol to disulfide conversion. (A) Rates of the bt-to-ds conversion were analyzed by HPLC, with tryptophan as an internal control. Representative HPLC results for peptide 2 with alanine (Λ = A) as the guest residue are shown. (B) Plots of bt-to-ds conversion for different guest residues. (C) CD spectra of sequences with different guest residues illustrate that the helical content of most sequences, with the exception of sequences with Λ = G, P, and D, is identical. The CD studies were performed with 50 μM peptides in 5 mM KF, pH 7.3.

Fig. 4.

Results of metadynamics simulation to evaluate barrier to the formation of an i to i + 4 hydrogen bond in a model peptide (AcAΛA-NHMe) as a function of different guest residues. (A and B) Two-dimensional free-energy surfaces for Λ = alanine and proline are shown with the others included in SI Text. The Ramachandran plots of all residues show low energy basins for the α, β, and polyproline II (PPII) dihedral space with the exception of proline, where the PPII and α spaces dominate. (C) Weighted populations of all low-energy basins corresponding to the α, β, and PPII dihedral angles. (D) The activation energy barrier between the lowest energy region corresponding to the PPII space and α-helical dihedral angles.