Hydroxylation-induced Stabilization of the Collagen Triple Helix

Abstract

The collagen triple helix is one of the most abundant protein motifs in animals. The structural motif of collagen is the triple helix formed by the repeated sequence of -Gly-Xaa-Yaa-. Previous reports showed that H-(Pro-4(R)Hyp-Gly) 10 -OH (where '4(R)Hyp' is (2S,4R)-4-hydroxyproline) forms a trimeric structure, whereas H-(4(R)Hyp-Pro-Gly) 10 -OH does not form a triple helix. Compared with H-(Pro-Pro-Gly) 10 -OH, the melting temperature of H-(Pro-4(R)Hyp-Gly) 10 -OH is higher, suggesting that 4(R)Hyp in the Yaa position has a stabilizing effect. The inability of triple helix formation of H-(4(R)Hyp-Pro-Gly) 10 -OH has been explained by a stereoelectronic effect, but the details are unknown. In this study, we synthesized a peptide that contains 4(R)Hyp in both the Xaa and the Yaa positions, that is, Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 and compared it to Ac-(GlyPro-4(R)Hyp) 10 -NH 2 , and Ac-(Gly-4(R)Hyp-Pro) 10 -NH 2 . Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 showed a polyproline II-like circular dichroic spectrum in water. The thermal transition temperatures measured by circular dichroism and differential scanning calorimetry were slightly higher than the values measured for Ac-(Gly-Pro-4(R)Hyp) 10 -NH 2 under the same conditions. For Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 , the calorimetric and the van't Hoff transition enthalpy ⌬H were significantly smaller than that of Ac-(Gly-Pro-4(R)Hyp) 10 -NH 2 . We postulate that the denatured states of the two peptides are significantly different, with Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 forming a more polyproline II-like structure instead of a random coil. Two-dimensional nuclear Overhauser effect spectroscopy suggests that the triple helical structure of Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 is more flexible than that of Ac-(Gly-Pro-4(R)Hyp) 10 -NH 2 . This is confirmed by the kinetics of amide 1 H exchange with solvent deuterium of Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 , which is faster than that of Ac-(Gly-Pro-4(R)Hyp) 10 -NH 2 . The higher transition temperature of Ac-(Gly-4(R)Hyp-4(R)Hyp) 10 -NH 2 , can be explained by the higher trans/cis ratio of the Gly-4(R)Hyp peptide bonds than that of the Gly-Pro bonds, and this ratio compensates for the weaker interchain hydrogen bonds.The collagen triple helix is one of the most abundant protein motifs in animals. Each of the three polypeptide chains forms a polyproline-II like structure, and these structures twist around each other to form a right-handed superhelix (1, 2). To form this structure, a sequence with Gly in every third position is required. The general sequence is -Gly-Xaa-Yaa-, and the Xaa and Yaa positions contain a high content of proline and hydroxyproline. Twenty-seven types of collagens and more than fifteen proteins that form a triple helix, but are not named collagens such as collectins, ficolins, and scavenger receptors (3-5), occur in humans. In vertebrates, most of the proline residues in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence are post-translationally modified to 4(R)Hyp 1 by prolyl 4-hydroxylase (EC 1.14.1...

Prolyl 3-Hydroxylase 1, Enzyme Characterization and Identification of a Novel Family of Enzymes

“…In contrast the role of 3(S)-hydroxyproline in collagens is not well understood. However, recent studies have shown that 3(S)-hydroxyproline destabilizes the triple helix and that peptides with the sequence Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH 2 do not form a triple helix (37,38). These studies have suggested the importance of 3(S)Hyp in modulating the stability of basement membrane collagens to allow for the meshwork-like structures they form.…”

4‐Chloroprolines: Synthesis, conformational analysis, and effect on the collagen triple helix

“…[22][23][24] CRPs with Hyp in the Yaa position are more stable than those with Pro, but Hyp in the Xaa position prevents triple-helix formation when the Yaa amino acid is Pro. [24][25][26][27][28][29] In the last decade, we have demonstrated that the stability (or instability) conferred by Hyp derives from the manifestation of previously unappreciated stereoelectronic effects. We did so by installing functional groups at C γ that mediate such effects, as well as reciprocal steric effects ( Figure 1).…”

The Crystal Structure of a Collagen-like Polypeptide with 3(S)-Hydroxyproline Residues in the Xaa Position Forms a Standard 7/2 Collagen Triple Helix

“…Indeed, it was thought that a triple helix with 3(S)Hyp residues in the Xaa position would be more stable than one with Pro residues at this position, as Pro does not favor one pucker type (up or down) over the other. Thus, it was a surprise when it was found that peptides with sequences containing long stretches of (3(S)Hyp)-(4(R)Hyp)-Gly do not form stable triple helices (33). However, when Raines and co-workers (20) examined this issue via host guest peptides, specifically, with (Pro-4(R)Hyp-Gly) 3 -3(S)Hyp-4(R)Hyp-Gly-(Pro-4(R)Hyp-Gly) 3 , they found that the presence of 3(S)Hyp residues embedded in the Xaa position of this peptide only decreased the transition midpoint temperature (T m ) value of the triple helix formation by 3°C compared with a peptide with a Pro in the corresponding position.…”