Signature of n→π* interactions in α-helices

Abstract

The oxygen of a peptide bond has two lone pairs of electrons. One of these lone pairs is poised to interact with the electron-deficient carbon of the subsequent peptide bond in the chain. Any partial covalency that results from this n→π* interaction should induce pyramidalization of the carbon (C′_i) toward the oxygen (O_i−1). We searched for such pyramidalization in 14 peptides that contain both α- and β-amino acid residues and that assume a helical structure. We found that the α-amino acid residues, which adopt the main chain dihedral angles of an α-helix, display dramatic pyramidalization but the β-amino acid residues do not. Thus, we conclude that O_i−1 and C′_i are linked by a partial covalent bond in α-helices. This finding has important ramifications for the folding and conformational stability of α-helices in isolation and in proteins.

Introduction

Electron delocalization is the source of partial covalency in many noncovalent interactions. For example, the partial covalency in a hydrogen bond stems from delocalization of the lone pair of electrons (n) of the hydrogen bond acceptor over the antibonding orbital (σ*) of the hydrogen bond donor.^1–6 We have discovered another noncovalent interaction, termed the n→π* interaction, with partial covalency.^7–11 In this interaction, the partial covalency arises due to overlap of the electron pair (n) of a donor group with the antibonding orbital (π*) of a carbonyl group.^12–18 In common protein secondary structures ^{11 19} and peptoids, ^{20 21} the electron-pair donor is a proximal carbonyl oxygen. This interaction is the basis of many protein–ligand interactions ²² and is reminiscent of the Bürgi–Dunitz trajectory for nucleophilic additions to carbonyl groups.²³

The partial covalency of the n→π* interaction should give rise to a distinctive structural signature. Specifically, an n→π* interaction, analogous to the approach of a nucleophile to a carbonyl group, ²³ should engender pyramidalization of the acceptor carbon of the carbonyl group.¹⁰ In this pyramidalization, the acceptor carbon is displaced toward the donor group and away from the plane formed by its three pendant atoms (Fig. 1). Pyramidalization can be detectable in high-resolution crystal structures. Nonetheless, the magnitude of the pyramidalization is small, and its origin can be attributed to other forces, such as those involved in the formation of the crystal lattice.^{24 25} Here, we use α/β-peptides to search for the existence of this definitive signature for the n→π* interaction in residues with an α-helical conformation. Our findings provide new insight on this most renowned and common secondary structures.

Figure 1.

Pyramidalization of main-chain carbonyl groups due to the n→π* interaction showing the definition of distances: d and Δ and angles: θ and θ.

Results and Discussion

In an α-helix, the lone pair (n) of the oxygen (O_i−1) of the amide carbonyl group at position i − 1 overlaps with the antibonding orbital (π*) of the amide carbonyl group (C′_i=O_i) at position i. This n→π* electron delocalization induces a short contact between these two carbonyl groups. It follows that the n→π* interaction could engender pyramidalization of the carbon of the acceptor carbonyl group, which would be observable in the high-resolution crystal structures of α-helices.

To establish that any observed pyramidalization arises from an n→π* interaction, an internal control is needed, wherein pyramidalization is absent when the n→π* interaction is absent. Gellman and coworkers ^26–30 have determined crystal structures for 14 helical peptides containing both α- and β-amino acid residues (see Supporting Information). These structures were solved by direct methods rather than by experimental phasing or molecular replacement, and the atomic coordinates were deposited in the Cambridge Crystallographic Data Centre (CCDC). In these structures, the α-amino acid residues adopt the torsional angles φ (C′_i−1–N_i–C_i^α–C′_i) and ψ (N_i–C_i^α–C′_i–N_i+1) that characterize an α-helix (Fig. 2).^{29 30} A close examination of these crystal structures revealed that carbonyl groups flanking the α-amino acid residues exhibited a C′_i−1 =O_i−1···C′_i distance of d < 3.22 Å, but analogous carbonyl groups flanking the β-amino acid residues had d > 3.22 Å, where 3.22 Å is the sum of the van der Waals radii of oxygen and carbon. The larger values of d for the β-amino acid residues stem from the additional carbon in their main chain. Hence, the carbonyl groups of the α-amino acid residues should experience a stronger n→π* interaction than the carbonyl groups of the β-amino acid residues. Accordingly, we reasoned that the carbonyl groups of the α-amino acid residues could likewise exhibit greater pyramidalization.

Figure 2.

Ramachandran plot of α-amino acid residues in the 14 (αβ)_n peptides analyzed herein. The ball-and-stick diagram depicts the structure with CCDC refcode OGAVAU.

The parameter θ reports on the extent of carbonyl pyramidalization (Fig. 1).¹⁰ In an α-helix, a value of θ > 0 indicates that C′_i is closer to O_i−1 (and to the center of the α-helix) than the plane defined by O_i, C_i^α, and N_i+1. Conversely, a value of θ < 0 indicates that C′_i is farther from O_i−1 than that plane.

An examination of 14 crystal structures of oligopeptides with alternating α- and β-amino acid residues revealed that the acceptor carbonyl groups of the α-amino acid residues exhibit larger pyramidalization than those of the β-amino acid residues. Moreover, that pyramidalization has θ > 0, which is consistent with the partial covalency between O_i−1 and C′_i. The mean value of θ for α-amino acid residues was (4.0° ± 1.1°), whereas that for β-amino acid residues was −(1.2° ± 1.9°) [Fig. 3(A)]. The first and the last residues were not included in this calculation and are also not depicted in Figures 2 and 3(A).

Figure 3.

Pyramidalization in α/β peptides. The parameter θ is a measure of pyramidalization (Fig. 1). Data are from crystal structures in the CCDC.^26–30 •, α-amino acid residue; ○, β-amino acid residue. A: (αβ)_n (all 14); CCDC refcode OGATAS, CAXRID, OGASOF, OGASUL, OGATEW, OGATIA, COVFUP, OGATOG, OGATUM, OGAVAU, OGAVEY, OGAVIC, OGAVOI, and COVGAW. B: (αβ)_n (subset); CCDC refcode COVFUP, OGATUM, and OGAVAU. C: (α₂β)_n; CCDC refcode PUCCIA and PUCCOG (two asymmetric units). D: (αβ₂)_n; CCDC refcode PUCCUM, PUCDEX, and PUCDUN.

It is noteworthy that the β-amino acid residues tend to have θ < 0. This inversion can be attributed to Pauli repulsion between the lone pair of the donor oxygen and the π orbital of the carbonyl group (Fig. 4).³¹ The slight negative pyramidalization of the acceptor carbonyl carbon obviates this Pauli repulsion. Similar closed shell repulsions had been invoked previously to explain small carbonyl pyramidalization.^{24 25} We also note that the oxygen donor for a β-amino acid residue blocks the si face of the acceptor carbonyl group, as the donor oxygen is close to the carbon acceptor (d ≈ 3.5–4.0 Å) but not close enough to induce a positive pyramidalization. The re face of the carbonyl group is exposed to short contacts from solvent and other molecules in the crystal lattice that could induce a small negative pyramidalization.

Figure 4.

Typical α- and β-amino acid residues in α/β peptides. A: Favorable overlap between n- and the π*-orbital in an α-amino acid residue. B: Unfavorable overlap between n- and the π-orbital in a β-amino acid residue. Orbital images were obtained using computational methods described previously ¹¹ on residues in CCDC refcode OGAVAU capped with acetyl and N-methylamino groups.

Quite strikingly, the carbonyl pyramidalization follows a distinct sawtooth pattern in helical peptides with alternating α- and β-amino acid residues [Fig. 3(B)]. To ascertain that the sawtooth pattern for alternating α- and β-amino acid residues was dependent on residue type, we analyzed crystal structures of helical peptides with α₂β- and αβ₂-amino acids as repeating motifs. In all of these sequences, we found that the pyramidalization pattern was indeed dependent on the type of residue [Fig. 3(C,D)]. The α-amino acid residues showed consistent positive pyramidalization, and the β-amino acid residues showed either negligible or slightly negative pyramidalization.

These findings lend strong credence to the existence of n→π* interaction in α-helices. Consistent with our findings, Mazzarella and coworkers reported carbonyl pyramidalization in α-helices of high-resolution protein crystal structures.³² Likewise, using average isotropic chemical shifts and X-ray crystallography, Lario and Vrielink ³³ reported that the π-electron clouds of carbonyl groups are more polarized in α-helices than that in β-sheets. In α-helices, the adjacent carbonyl groups are poised for a strong n→π* interaction, but they are too far apart for any significant n→π* interaction in β-sheets. Accordingly, the polarization of the π-bond, which is a natural outcome of the n→π* interaction, is apparent in α-helices but not β-sheets. Such polarization of the π-bond could strengthen the canonical i→i + 4 hydrogen bond by making the carbonyl oxygen a better hydrogen-bond acceptor.

The existence of the n→π* interaction in α-helices has bearing on their folding and conformational stability. The short contact effected by the n→π* interaction between adjacent carbonyl groups fortifies a compact structure that aligns the distal hydrogen-bond donor and acceptor groups for a strong hydrogen bond. In an α-helix, the s-rich carbonyl lone pair participates in i→i+4 hydrogen bond. The other lone pair, which is p-rich, is engaged in the n→π* interaction between adjacent carbonyl groups.¹¹ The n→π* interaction not only engages this lone pair for intramolecular association but also prevents it from participating in structure-disrupting hydrogen bonds with other molecules. In addition to contributing to conformational stability, the n→π* interaction could contribute to the folding of an α-helix. The nucleation of an α-helix involves the formation of its first turn, which is disfavored by both entropy and enthalpy.^34–36 The n→π* interaction, which operates between the adjacent carbonyl groups, can compensate for these energetic penalties.