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

Abstract

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.

Bioinspired photo-crosslinkable self-assembling peptides with pH-switchable “on–off” luminescence

“…Please do not adjust margins For instance, Hartgerink and his team reported the use of covalent capture of collagen mimetic peptides (CMPs), through the formation of isopeptide bonds between lysine and either aspartate or glutamate, using carboxylate activating reagents 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and Hydroxybenzotriazole (HOBt) [24,25]. By using a similar approach, our laboratory reported the design of covalent capture of SAPhydrogel using a one-pot in situ gelation system, based on EDC/Nhydroxysulfosuccinimide (sulfo-NHS) coupling, to readily cross-link LDLK12 molecules, in order to tune its biomechanics without affecting the spontaneous formation of β-sheet-containing nanofilaments [16].…”

Modulating the Stability of Collagen Triple Helices by Terminal Charged Residues

“…Previous studies have shown that the interchain cationic−anionic and interchain cation−π interactions can induce the formation of collagen heterotrimers, 19−24 and interstrand covalent bonds between cationic and anionic residue side chains can form upon mixing the CMPs to assist self-assembly. 25,26 These findings further indicate that the installed terminal residue will have its side chain oriented to the exterior of the triple helix, allowing us to examine the impacts of short-range side chain− terminus and long-range side-chain−side-chain interactions on the triple helix via our designed CMPs. Previous works had used the cationic and anionic residues in various side-chain lengths to show the length effects on the ion−pair interactions and the stability of short α-helices, β-sheets, and collagen triple helices.…”

Selective covalent capture of collagen triple helices with a minimal protecting group strategy

“…Alpha helical, 12 beta sheet [13][14][15][16][17] and polyproline type 2 secondary [18][19][20][21] structures have been prepared by this selfassembly followed by covalent capture approach.…”

“…Our group recently developed a covalent capture technique for collagen triple helices in which isopeptide bonds are formed between the side chains of lysine and aspartate or glutamate. [20][21][22] These engineered collagens were found to have improved thermal stability and an increased rate of folding while perfectly preserving the native triple helical fold of the assembled peptides.…”