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. 2022 Feb 17;13(9):2789-2796.
doi: 10.1039/d1sc06361h. eCollection 2022 Mar 2.

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

Le Tracy Yu  1 , Jeffrey D Hartgerink  1
Affiliations

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

Le Tracy Yu et al. Chem Sci. .

Abstract

Collagens and their most characteristic structural unit, the triple helix, play many critical roles in living systems which drive interest in preparing mimics of them. However, application of collagen mimetic helices is limited by poor thermal stability, slow rates of folding and poor equilibrium between monomer and trimer. Covalent capture of the self-assembled triple helix can solve these problems while preserving the native three-dimensional structure critical for biological function. Covalent capture takes advantage of strategically placed lysine and glutamate (or aspartate) residues which form stabilizing charge-pair interactions in the supramolecular helix and can subsequently be converted to isopeptide amide bonds under folded, aqueous conditions. While covalent capture is powerful, charge paired residues are frequently found in natural sequences which must be preserved to maintain biological function. Here we describe a minimal protecting group strategy to allow selective covalent capture of specific charge paired residues which leaves other charged residues unaltered. We investigate a series of side chain protecting groups for lysine and glutamate in model peptides for their ability to be deprotected easily and in high yield while maintaining (1) the solubility of the peptides in water, (2) the self-assembly and stability of the triple helix, and (3) the ability to covalently capture unprotected charge pairs. Optimized conditions are then illustrated in peptides derived from Pulmonary Surfactant protein A (SP-A). These covalently captured SP-A triple helices are found to have dramatically improved rates of folding and thermal stability while maintaining unmodified lysine-glutamate pairs in addition to other unmodified chemical functionality. The approach we illustrate allows for the covalent capture of collagen-like triple helices with virtually any sequence, composition or register. This dramatically broadens the utility of the covalent capture approach to the stabilization of biomimetic triple helices and thus also improves the utility of biomimetic collagens generally.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Illustration of salt-bridges in collagen triple helices between the side chains of Lys and Glu. (A) Axial interaction. The figure is generated from PDB 3t4f. (B) Isopeptide bond formation by covalently capturing the axial salt-bridge. Figure is generated from PDB 6vzx. (C) Lateral interaction. (D) Isopeptide bond formation by covalently capturing the lateral salt-bridge. Models and figures were generated with PyMOL 2.5.1.
Scheme 1
Scheme 1. Selective covalent capture of collagen triple helix with protecting groups. The designed triple helices are expected to have a canonical registration. “O” represents (4R)-hydroxyproline. Blue and red colors represent positively and negatively charged amino acid residues, respectively. Yellow highlights the residues that will be protected by Fmoc-orthogonal protecting groups, specifically ivDde on the side chain of lysine, OAll, OBzl, and ODmab on the side chain of glutamic acid. (A and B) are template triple helices with different protecting schemes (Table S1†). (C and D) are modified peptide sequences from protein rat SP-A. Triple helix (C and D) have 6 and 3 iso-peptide bond formations, respectively. The sequences were obtained from universal protein knowledgebase.
Fig. 2
Fig. 2. Circular dichroism of template collagen triple helices with protecting groups. (a) The melting (denaturation) curves measured at the wavelength of 225 nm. (b) The first-order derivative curves of the corresponding melting curves.
Fig. 3
Fig. 3. Hydrolysis kinetics of allyl and benzyl esters from covalently captured collagen triple helices ccKGE(OAll) and ccKGE(OBzl). (a) Allyl ester hydrolysis with 80 mM K2CO3 at different temperatures. (b) Benzyl ester hydrolysis with 80 mM K2CO3 at different temperatures. (c) Benzyl ester hydrolysis at 45 °C with K2CO3 solutions of different basicity.
Fig. 4
Fig. 4. ESI Mass spectrum of covalently captured KGE template triple helices. The triple helices were removed of (a) allyl ester, (b) benzyl ester and (c) ivDde protecting groups.
Fig. 5
Fig. 5. CD results of KGE triple helices. (a) CD melt and refold curves of supramolecular KGE triple helix. (b) The first-order derivative curves of the melting curves in (a). (c) CD melting and refolding curves of covalently captured KGE triple helices from benzyl protecting group removal. (d) The first-order derivative curves of the melting curves in (c).
Fig. 6
Fig. 6. ESI mass spectra of covalently captured SP-A branch triple helices. (a) Covalently captured SP-A1 triple helix after ivDde deprotection. (b) Covalently captured SP-A2 triple helix after ivDe deprotection.
Fig. 7
Fig. 7. CD results of SP-A triple helices. (a) CD melt and refolding curves of supramolecular SP-A1 triple helices. (b) first-order derivative of the melting curves in part (a). (c) CD melt and refolding curves of supramolecular SP-A2 triple helices. (d) first-order derivative of the melting curves in part (d). (e) CD melt and refolding curves of covalently captured SP-A1 triple helix. (f) first-order derivative of the melting curves in part e.g. CD melt and refolding curves of covalently captured SP-A2 triple helix. (h) first-order derivative of the melting curves in part (g).
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