Abstract:

<p>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&#39;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&#39; side chain bulkiness influenced stability only at the Xaa position and not the Yaa position. Additionally, only -isomers of chiral N-C-branched N-Glys were compatible with triple helical folding, presenting different backbone conformations and accessible rotamers based on their position. At the Yaa position, ()-N-(1-phenylethyl)-Gly (Nspe) stabilized the triple helix better than all other residues except Hyp through newly discovered intrachain CH&middot;&middot;&middot;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.</p>