Stabilization of the Collagen Triple Helix by O-Methylation of Hydroxyproline Residues

Collagen is the most abundant protein in animals, including humans. The prevalent (2S,4R)-4-hydroxyproline (Hyp) residues of collagen are known to confer great stability upon its triple-helical conformation. The basis for that stability has been attributed to hydration of the pendant hydroxyl groups. Here, that attribution is shown to be incorrect. Replacement of the natural Hyp residues with synthetic (2S,4R)-4-methoxyproline (Mop) residues is shown by circular dichroism spectroscopy and differential scanning calorimetry to increase the conformational stability of the collagen triple helix. The thermodynamic parameters indicate that, as expcted, O- methylation decreases the hydration of the triple helix. Apparently, hydration of Hyp residues is deleterious, rather than advantageous, to the collagen triple helix. The crystal structure of Ac-Mop-OMe reveals the manifestation of two stereoelectronic effects: a gauche effect and an n rarr pi interaction, which preorganize the main-chain atoms properly for triple-helix formation. Thus, the conformational stability conferred upon the collagen triple helix by O-methylation provides strong evidence that the hydroxyl group of Hyp acts primarily through stereoelectronic effects and that its hydration provides no benefit. This information could have practical utility, as Mop could be prepared in situ by the O-methylation of Hyp residues in natural collagen. Such a semisynthetic collagen could have superior properties as a biomaterial.