Recent Advances in Collagen Mimetic Peptide Structure and Design.

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.

Cation-π Interactions and Their Role in Assembling Collagen Triple Helices.

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 ABC-type 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.

Covalent Capture of Collagen Triple Helices Using Lysine-Aspartate and Lysine-Glutamate Pairs.

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.

Heterotrimeric collagen helix with high specificity of assembly results in a rapid rate of folding.

The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood. Here we compare the stabilities, specificities and folding rates of three heterotrimeric collagen mimics designed through a computationally assisted approach. The crystal structure of one ABC-type heterotrimer verified a well-controlled composition and register and elucidated the geometry of pairwise cation-π and axial and lateral salt bridges in the assembly. This collagen heterotrimer folds much faster (hours versus days) than comparable, well-designed systems. Circular dichroism and NMR data suggest the folding is frustrated by unproductive, competing heterotrimer species and these species must unwind before refolding into the thermodynamically favoured assembly. The heterotrimeric collagen folding rate is inhibited by the introduction of preformed competing triple-helical assemblies, which suggests that slow heterotrimer folding kinetics are dominated by the frustration of the energy landscape caused by competing triple helices.

Predicting the stability of homotrimeric and heterotrimeric collagen helices.

Robust methods for predicting thermal stabilities of collagen triple helices are critical for understanding natural structure and stability in the collagen family of proteins and also for designing synthetic peptides mimicking these essential proteins. In this work, we determine the relative stability imparted on the collagen triple helix by single amino acids and interactions between amino acid pairs. Using this analysis, we create a comprehensive algorithm, SCEPTTr, for predicting melting temperatures of synthetic triple helices. Critically, our algorithm is compatible with every natural amino acid, can evaluate both homotrimers and heterotrimers, and accounts for all possible helix compositions and registers, including non-canonically staggered helices. We test and optimize our algorithm against 431 published collagen triple helices to demonstrate the quality of our predictive system. Finally, we use this algorithm to successfully guide the design of an ABC heterotrimer possessing high assembly specificity.

Covalent Capture of a Heterotrimeric Collagen Helix.

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.