Molecular foundations of collagen triple helical assembly: the central role of prolyl-4-hydroxylation

Collagen, the principal structural protein of the extracellular matrix, has been pivotal in the evolution of multicellular life. Among its posttranslational modifications (PTMs), prolyl-4-hydroxylation stands out as the most conserved and functionally critical. This hydroxylation not only facilitates polyproline II-like helicity in single collagen strands but also underpins the stability and integrity of the triple helix. In this review, we summarize the molecular foundations of collagen assembly, emphasizing both thermodynamic and stereo-electronic contributions of 4-hydroxyproline (4-Hyp). Insights from collagen-mimetic peptides are highlighted to elucidate main-chain torsional preferences, the positional dependence of proline ring puckering, and the correlation between 4-Hyp and enhanced thermal stability. Parallel discussions explore stereo-electronic effects revealed through quantum chemical studies, particularly charge-transfer interactions that modulate pyrrolidine ring conformation and peptide bond geometry. Finally, we summarize advances from periodic quantum mechanical calculations that quantify interstrand binding energies across different tripeptide motifs. Together, these findings provide a molecular-level perspective on how prolyl-4-hydroxylation has shaped the unique stability and evolution of collagenous helicity.