We have shown recently that glycosylation of threonine in the peptide Ac-(Gly-Pro-Thr) 10 -NH 2 with ␤-D-galactose induces the formation of a collagen triple helix, whereas the nonglycosylated peptide does not. In this report, we present evidence that a collagen triple helix can also be formed in the Ac-(Gly-Pro-Thr) 10 -NH 2 peptide, if the proline (Pro) in the Xaa position is replaced with 4-transhydroxyproline (Hyp). Furthermore, replacement of Pro with Hyp in the sequence

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... -Gal)) 10 -NH 2 increases the T m of the triple helix by 15.7°C. It is generally believed that Hyp in the Xaa position destabilizes the triple helix because (Pro-Pro-Gly) 10 and (Pro-Hyp-Gly) 10 form stable triple helices but the peptide (Hyp-Pro-Gly) 10 does not. Our data suggest that the destabilizing effect of Hyp relative to Pro in the Xaa position is only true in the case of (Hyp-Pro-Gly) 10 . Increasing concentrations of galactose in the solvent stabilize the triple helix of Ac-(Gly-Hyp-Thr) 10 -NH 2 but to a much lesser extent than that achieved by covalently linked galactose. The data explain some of the forces governing the stability of the annelid/ vestimentiferan cuticle collagens.