The peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 and acetyl-(Gly-Pro-3(S)Hyp)10-NH2 do not form a collagen triple helix

Hydroxylation of proline residues in the Yaa position of the Gly-Xaa-Yaa repeated sequence to 4(R)-hydroxyproline is essential for the formation of the collagen triple helix. A small number of 3(S)-hydroyxyproline residues are present in most collagens in the Xaa position. Neither the structural nor a biological role is known for 3(S)-hydroxyproline. To characterize the structural role of 3(S)-hydroxyproline, the peptide Ac-(Gly-3(S)Hyp4(R)Hyp)(10)-NH2 was synthesized and analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and H-1 nuclear magnetic resonance spectroscopy. At 4degreesC in water the circular dichroism spectrum indicates that this peptide was in a polyproline-II-like secondary structure with a positive peak at 225 nm similar to Ac-(Gly-Pro4(R)Hyp)(10)-NH2. The positive peak at 225 nm almost linearly decreases with increasing temperature to 95degreesC without an obvious transition. Although the peptide Ac(Gly-Pro-4(R)Hyp)(10)-NH2 forms a trimer at 10degreesC, sedimentation equilibrium experiments indicate that Ac-(Gly3(S)Hyp-4(R)Hyp)(10)-NH2 is a monomer in water at 7degreesC. To study the role of 3(S)-hydroxyproline in the Yaa position, we synthesized Ac-(Gly-Pro-3(S)Hyp)(10)-NH2. This peptide also does not form a triple helix in water. H-1 Nuclear magnetic resonance spectroscopy data (including line widths and nuclear Overhauser effects) are entirely consistent, with neither Ac-(Gly-3(S)Hyp-4( R)Hyp)(10)-NH2 nor Ac-(Gly-Pro-3( S)Hyp)(10)-NH2 forming a triple helix in water. Therefore 3(S)-hydroxyproline destabilizes the collagen triple helix in either position. In contrast, when 3(S)-hydroxyproline is inserted as a guest in the highly stable-Gly-Pro-4(R)Hyp-repeated host sequence, Ac-(Gly-Pro-4(R)Hyp)(3)-Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)Hyp)(4)-Gly-Gly-NH2 forms as stable a trimer (T-m=49.6degreesC) as Ac-( Gly-Pro-4(R)Hyp)(8)-Gly-Gly-NH2 (T-m=48.9degreesC). Given that Ac-( Gly-Pro- 4(R)Hyp)(3)-Gly-4(R)Hyp-Pro-(Gly-Pro4(R)Hyp)(4)-Gly-Gly-NH2 forms a triple helix nearly as stable as the above two peptides (T-m=45.0degreesC) and the knowledge that Ac-(Gly-4(R)Hyp-Pro)(10)-NH2 does not form a triple helix, we conclude that the host environment dominates the structure of host-guest peptides and that these peptides are not necessarily accurate predictors of triple helical stability.