Hydroxylation-induced stabilization of the collagen triple helix. Further characterization of peptides with 4(R)-hydroxyproline in the Xaa position

4(R)-Hydroxyproline in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence of collagen plays a crucial role in the stability of the triple helix. Since the peptide (4(R)-Hyp-Pro-Gly)10 does not form a triple helix, it was generally believed that polypeptides with a -Gly-4(R)-Hyp-Yaa-repeated sequence do not form a triple helix. Recently, we found that acetyl-(Gly-4(R)-Hyp-Thr)10-NH2 forms a triple helix in aqueous solutions. To further study the role of 4(R)-hydroxyproline in the Xaa position, we made a series of acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptides where Yaa was alanine, serine, valine, and allo-threonine. We previously hypothesized that the hydroxyl group of threonine might form a hydrogen bond to the hydroxyl group of 4(R)hydroxyproline. In water, only the threonine- and the valine-containing peptides were triple helical. The remaining peptides did not form a triple helix in water. In 1,2- and in 1,3-propanediol at 4 degrees C, all the soluble peptides were triple helical. From the transition temperature of the triple helices, it was found that among the examined residues, threonine was the most stable residue in the acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptide. The transition temperatures of the valine- and allo-threonine-containing peptides were 10 degrees lower than those of the threonine peptide. Surprisingly, the serine-containing peptide was the least stable. These results indicate that the stability of these peptides depends on the presence of a methyl group as well as the hydroxyl group and that the stereo configuration of the two groups is essential for the stability. In the threonine peptide, we hypothesize that the methyl group shields the interchain hydrogen bond between the glycine and the Xaa residue from water and that the hydroxyl groups of threonine and 4(R)hydroxyproline can form direct or water-mediated hydrogen bonds.