Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly

Abstract

The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an alpha-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of alpha- and beta-amino acid residues but an invariant side-chain sequence. In the pure alpha-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of alpha/beta-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the alpha prototype. There is a loss in helix bundle stability upon beta-residue incorporation; however, stability of the quaternary structure is not a simple function of beta-residue content. We find that cyclically constrained beta-amino acid residues can stabilize the folds of alpha/beta-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.

Fig. 1.

Chemical structures and helical wheel diagrams of 1–9. (A) Primary sequences of α-peptide 1 and α/β-peptides 2–9, sorted according to α/β backbone pattern. Bold letters indicate hydrophobic core residues in the GCN4-pLI sequence. Colored circles indicate sequence positions occupied by β-residues, cyan for β³-residues and orange for cyclic β-residues. (B) Helical wheel diagram of 1 with hydrophobic core residues indicated. (C) Structures of an α-amino acid, a β³-amino acid, and the cyclic β-amino acids ACPC (X) and APC (Z). (D) Helical wheel diagrams of the α/β residue patterns of 2–9 based on a heptad repeat. Each circle represents a residue and is colored by residue type, yellow for α-residues, cyan for β³-residues, and orange for cyclic β-residues. Bold circles indicate hydrophobic core positions.

Fig. 2.

Comparison of the GCN4-pLI side chain sequence on four different backbone patterns. (A) Helix bundle quaternary structures of each derivative with yellow and blue indicating α- and β³-residues, respectively. (B) Helical secondary structures of each α/β-peptide 2–4 (red) compared with that of α-peptide 1 (yellow); the overlays and accompanying RMSD values are based on C_α atoms in shared α-residues. The coordinates for 1 (PDB: 1GCL) (21) and 2 (PDB: 2OXK) (14) were obtained from previously published structures.

Fig. 3.

CD spectra of 1–9. (A) CD spectra for GCN4-pLI α-peptide 1 and α/β-peptides 2–6 generated from simple α → β³ substitution. (B) CD spectra for GCN4-pLI α/β-peptide derivatives bearing cyclic β-residues. The colors of the spectra in B match the corresponding acyclic β-residue derivatives in A. All spectra were acquired for 100 μM α-peptide or α/β-peptide in 10 mM NaOAc (pH 4.6).

Fig. 4.

Comparison of the crystal structures of α/β-peptides 4 and 7. (A) Four-helix bundle quaternary structure of α/β-peptide 7. (B) Overlay of the helical backbone of 7 (pink) with that of 4 (blue). (C–E) Comparison of an acyclic β³-residue in 4 to the cyclic β-residue at the same position in 7: β³-hGlu₁₀ in 4 (C); β-ACPC₁₀ in 7 (D); overlay of C and D (E). (F and G) Top view of the hydrophobic core packing at residues 22 and 23 in 4 (F) and 7 (G).