Structural and Positional Effects of Peptoid Residues on Triple Helix Stability

The collagen triple helix, composed of three protein strands with a repeating GlyXaaYaa sequence, achieves maximum stability with proline (Pro) and hydroxyproline (Hyp) at the Xaa and Yaa positions. Previously, we demonstrated that peptoid residues (N-substituted glycines, N-Glys) at the Xaa position significantly enhance the triple helix stability. Here, we show that N-Glys at the Yaa position also stabilize the triple helix, with stability influenced by the N-Gly's position and side chain structure. Over 22 N-Glys with various natural and unnatural side chains were investigated using CD spectroscopy, X-ray crystallography, and computational simulations. The results show that N-Glys at the Yaa position support triple helical folding and are more stabilizing than their corresponding amino acids but less so than Hyp due to suboptimal ϕ-ψ angles. Metadynamics simulations revealed that N-Glys at the Yaa position have a broader conformational space due to minimal steric hindrance from neighboring glycine residues. N-Glys' side chain bulkiness influenced stability only at the Xaa position and not the Yaa position. Additionally, only S-isomers of chiral N^α-C^β-branched N-Glys were compatible with triple helical folding, presenting different backbone conformations and accessible rotamers based on their position. At the Yaa position, (S)-N-(1-phenylethyl)-Gly (Nspe) stabilized the triple helix better than all other residues except Hyp through newly discovered intrachain CH···π interactions. This research deepens our understanding of triple helical folding and introduces new strategies for designing stable collagen mimetic peptides with diverse side chains, expanding potential applications in biomedicine and biomaterials.