Collagen’s structural integrity derives from its repeating Gly–Xaa–Yaa motif and right-handed triple helical architecture, wherein proline (Pro) and 4(R)-hydroxyproline (Hyp) at the Xaa and Yaa positions, respectively, impose conformational constraints that promote folding. While the cyclic rigidity and stereoelectronic properties of these residues are well-established contributors to triple helix stability,[1] recent studies with peptoid residues at the Xaa position suggest that tertiary amide geometry may also play a critical role, independent of ring structure.[2] Here, we employ a host–guest collagen-mimetic peptide (CMP) system, Ac-(GlyProHyp)₃-Gly–Xaa–Hyp-(GlyProHyp)₃-NH₂, to systematically investigate the influence of tertiary amide conformation and Cα-substitution at the Xaa position. A diverse panel of N-methyl amino acids, N-substituted alanines, and other tertiary amide–containing residues with (S)-Cα chirality was synthesized and evaluated alongside canonical amino acids and peptoid residues for triple helix thermal stability and refolding kinetics. Notably, N-substituted alanines bearing benzylic side chains, such as Nphe-Ala and Ntyr-Ala, not only exceed Pro in stabilizing the triple helix but also significantly accelerate refolding. In contrast, increasing the size of the Cα-substituent beyond methyl markedly disrupts triple helix formation, likely due to steric clashes with the adjacent Hyp residue. X-ray crystallography and molecular dynamics simulations reveal that benzylic groups at the amide nitrogen promote inter-strand CH/π interactions, whereas analogous substitution at Cα leads to destabilizing intra-strand contacts. These findings delineate the distinct structural roles of amide nitrogen and Cα-substitution in modulating collagen folding and establish design principles for CMPs with enhanced thermodynamic and kinetic properties.
Keywords: Collagen mimetic peptide, Circular Dichroism, Thermodynamic Stability, Folding Kinetics