In-depth correlation analysis demonstrates that 4-hydroxyproline at the Yaa position of Gly-Xaa-Yaa repeats dominantly stabilizes collagen triple helix

There is a general consensus that collagen stability is largely maintained by Pro and its major hydroxylated form, 4-hydroxyproline (4Hyp). However, positional difference in their stabilizing effect at the Xaa or Yaa position of collagenous Gly-Xaa-Yaa sequences has remained inconclusive. Here, we position-specifically evaluated the correlation of imino acid contents to denaturation temperature (T(d)) of collagen among various vertebrate and invertebrate species, using a recently developed LC–MS methodology. 4Hyp at the Yaa position showed the highest positive correlation with T(d), followed by Pro at the Xaa position, which was even further increased by excluding invertebrates. We confirmed that Gly-Pro-4Hyp liberated after bacterial collagenase digestion was highly positively correlated with T(d). Furthermore, other tripeptides with Yaa position 4Hyp also had comparable positive correlation, excepting negative correlation of Gly-Gly-4Hyp, while tripeptides with Xaa position Pro did not. These data provide evidence that 4Hyp dominantly contributes to thermal stability of collagen depending on its sequence position, especially in vertebrates.