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.