No abstract

Stabilizing the three-dimensional structure of supramolecular materials can be accomplished through covalent capture of the assembled system. The lysine-aspartate charge pairs designed to direct the self-assembly of a collagen triple helix were subsequently used to covalently capture the helix through proximity-directed amide bond formation using EDC/HOBT activation. The triple helix thus stabilized maintains its folded structure and can now be used for applications previously inaccessible due to problematic folding equilibria.

Collagen mimetic peptides (CMPs) fold into a polyproline type II triple helix, allowing the study of the structure and function (or misfunction) of the collagen family of proteins. This Perspective will focus on recent developments in the use of CMPs toward understanding the structure and controlling the stability of the triple helix. Triple helix assembly is influenced by various factors, including the single amino acid propensity for the triple helix fold, pairwise interactions between these amino acids, and long-range effects observed across the helix, such as bend, twist, and fraying. Important progress in creating a comprehensive and predictive understanding of these factors for peptides with exclusively natural amino acids has been made. In contrast, several groups have successfully developed unnatural amino acids that are engineered to stabilize the triple helical structure. A third approach to controlling the triple helical structure includes covalent cross-linking of the triple helix to stabilize the assembly, which eliminates the problematic equilibrium of unfolding into monomers and enforces compositional control. Advances in all these areas have resulted in significant improvements to our understanding and control of this important class of protein, allowing for the design and application of more chemically complex and well-controlled collagen mimetic biomaterials.

Collagen mimetic peptides (CMPs) self-assemble into a triple helix reproducing the most fundamental aspect of the collagen structural hierarchy. They are therefore important for both further understanding this complex family of proteins and use in a wide range of biomaterials and biomedical applications. CMP self-assembly is complicated by a number of factors which limit the use of CMPs including their slow rate of folding, relatively poor monomer−trimer equilibrium, and the large number of competing species possible in heterotrimeric helices. All of these problems can be solved through the formation of isopeptide bonds between lysine and either aspartate or glutamate. These amino acids serve two purposes: they first direct self-assemble, allowing for composition and register control within the triple helix, and subsequently can be covalently linked, fixing the composition and register of the assembled structure without perturbing the triple helical conformation. This self-assembly and covalent capture are demonstrated here with four different triple helices. The formation of an isopeptide bond between lysine and glutamate (K−E) is shown to be a faster and higher yielding reaction than lysine with aspartate (K−D). Additionally, K−E amide bonds increase the thermal stability, improve the refolding capabilities, and enhance the triple helical structure as compared to K−E supramolecular interactions, observed by circular dichroism. In contrast, covalent capture of triple helices with K−D amide bonds occurs slower, and the captured triple helices do not have enhanced helical structure. The crystal structure of a triple helix captured through the formation of three K−E isopeptide bonds unequivocally demonstrates the connectivity of the amide bonds formed while also confirming the preservation of the canonical triple helix. The rate of reaction and yield for covalently captured K−E triple helices along with the excellent preservation of triple helical structure demonstrate that this approach can be used to effectively capture and stabilize this important biological motif for biological and biomedical applications.

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.