A Host−Guest Set of Triple-Helical Peptides: Stability of Gly-X-Y Triplets Containing Common Nonpolar Residues

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Abstract
Host−guest peptide sets have been useful in evaluating the propensity of different amino acids to adopt an α-helical or β-sheet form, and this concept is applied here to the triple-helical conformation. A set of host−guest peptides of the form acetyl-(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Glyc o n h 2 was designed to evaluate the contribution of an isolated Gly-X-Y triplet to triple-helix stability in a defined environment. Peptides were synthesized to include guest triplets with the X and Y positions occupied by the most common nonpolar residues found in collagen: Pro (X position) and Hyp (Y position); Ala; Leu, the most frequent hydrophobic residue; and Phe, the only commonly occurring aromatic residue. The guest triplets of the 12 peptides synthesized represent 35% of the sequence found in the α1 chain of type I collagen. All peptides formed stable triple-helical structures, and the peptides showed a range of thermal stabilities (Tm = 21−44 °C), depending on the identity of the guest triplet. Thermodynamic calculations indicate these peptides have a range of free energy values (ΔΔG = 9 kcal/mol) and suggest that favorable entropy is the dominant factor in increased stability. Replacement of Ala by Leu in the X position did not affect the thermal stability, while an Ala to Leu change in the Y position was destabilizing. These data provide experimental evidence that hydrophobic residues do not stabilize the triple helical conformation. Although Leu and Phe are found almost exclusively in the X position in collagens, peptides with Leu and Phe in the Y position formed stable triple-helices. This supports the hypothesis that the X positional preference of these residues relates to their increased potential for intermolecular hydrophobic interactions rather than their destabilization of the triple-helical molecule. These studies establish the utility of host−guest peptides in defining a scale of triple-helix propensities and in clarifying the interactions stabilizing the triple-helical conformation.