No abstract

Cation−π interactions play a significant role in the stabilization of globular proteins. However, their role in collagen triple helices is less well understood and they have rarely been used in de novo designed collagen mimetic systems. In this study, we analyze the stabilizing and destabilizing effects in pairwise amino acid interactions between cationic and aromatic residues in both axial and lateral sequential relationships. Thermal unfolding experiments demonstrated that only axial pairs are stabilizing, while the lateral pairs are uniformly destabilizing. Molecular dynamics simulations show that pairs with an axial relationship can achieve a near-ideal interaction distance, but pairs in a lateral relationship do not. Arginine−π systems were found to be more stabilizing than lysine−π and histidine−π. Arginine−π interactions were then studied in more chemically diverse ABCtype heterotrimeric helices, where arginine−tyrosine pairs were found to form the best helix. This work helps elucidate the role of cation−π interactions in triple helices and illustrates their utility in designing collagen mimetic peptides.

Collagen mimetic peptides (CMPs) are an excellent model to study the structural and biological properties of the extracellular matrix (ECM) due to ease of synthesis and variability in sequence. To ensure that synthetic materials accurately mimic the structure and function of natural collagen in the ECM, it is necessary to conserve the triple helix. However, CMP folding is subject to equilibrium, and frequently peptides exist in solution as both monomer and triple helix. Additionally, the stability of CMPs is highly dependent on peptide length and amino acid composition, leading to suboptimal performance. Here, we report the utility of covalent capture, a method to (a) direct the folding of a supramolecular triple helix and (b) form isopeptide bonds between the helix strands, in the design of an integrin-binding peptide with a GFOGER motif. Covalent capture effectively locked the triple helix and yielded a peptide with high thermal stability and a rapid folding rate. Compared to supramolecular triple helices bearing the same GFOGER-binding site, cell adhesion was substantially increased. In vitro assays using EDTA/Mg2+ and an anti-α2β1 antibody demonstrated the preservation of the high specificity of the binding event. This covalently captured integrin-binding peptide provides a template for the future design of bioactive ECM mimics, which can overcome limitations of supramolecular approaches for potential drug and biomaterial designs.