Positional Preferences of Ionizable Residues in Gly-X-YTriplets of the Collagen Triple-helix

Virginia C Chan,John A.M Ramshaw,Alan Kirkpatrick,Konrad Beck,Barbara Brodsky

doi:10.1074/jbc.272.50.31441

Virginia C Chan, John A.M Ramshaw + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.50.31441

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Abstract

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Y position of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Y peptides showed a wide thermal stability range (Tm = 30-45 degrees C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except when X is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.

Highlights

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding
Samples for circular dichroism (CD)2 spectroscopy were prepared at a concentration of 1 mg/ml, and pH measurements were made at 20 °C
The enthalpy was a stabilizing feature at all pH values, whereas the entropy was unfavorable compared with the host peptide containing only Gly-Pro-Hyp triplets. This investigation uses host-guest peptides to determine the propensity of ionizable residues for the triple-helix conformation in the different positions of the Gly-X-Y repeating unit

Summary

Introduction

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. One direct interchain N–H 1⁄7 1⁄7 OϭC hydrogen bond per Gly-X-Y triplet is formed in the triple-helix, leaving two carbonyl groups of each triplet available for hydrogen bonding These unsatisfied carbonyl groups are seen to be involved in a highly ordered hydration network in the crystal structure of a peptide with Gly-Pro-Hyp units [8, 12]. Ion-pairs do not appear to contribute a stabilizing effect in general but appear to be important when there is a basic residue in the X position and an Asp residue in the Y position

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