Abstract
The stability of the triple-helical structure of collagen is modulated by a delicate balance of effects including polypeptide backbone geometry, a buried hydrogen bond network, dispersive interfacial interactions, and subtle stereoelectronic effects. Although the different amino acid propensities for the Xaa and Yaa positions of collagen's repeating (Glycine–Xaa–Yaa) primary structure have been described, our understanding of the impact of incorporating aza-glycine (azGly) residues adjacent to varied Xaa and Yaa position residues has been limited to specific sequences. Here, we detail the impact of variation in the Xaa position adjacent to an azGly residue and compare these results to our study on the impact of the Yaa position. For the first time, we present a set of design rules for azGly-stabilized triple-helical collagen peptides, accounting for all canonical amino acids in the Xaa and Yaa positions adjacent to an azGly residue, and extend these rules using multiple azGly residues. To gain atomic level insight into these new rules we present two high-resolution crystal structures of collagen triple helices, with the first peptoid-containing collagen peptide structure. In conjunction with biophysical and computational data, we highlight the critical importance of preserving the triple helix geometry and protecting the hydrogen bonding network proximal to the azGly residue from solvent. Our results provide a set of design guidelines for azGly-stabilized triple-helical collagen peptides and fundamental insight into collagen structure and stability.
Highlights
Collagen, the most abundant protein in the human body, serves as a critical structural element in skin, blood vessels, muscles, bones, and cartilage.[1,2,3] Collagen proteins play key roles in several elds acting as pharmaceutical agents, matrices for tissue engineering, scaffolds for tissue regrowth, wound healing, and the development of biomaterials.[2,4,5,6] Collagen's structure involves the assembly of three polyproline II helices, with each individual chain consisting of a three residue repeat sequence with the general form (Glycine–Xaa– Yaa)
We established that incorporating an azGly residue adjacent to an Xaa position Pro residue resulted in an increase of its triple-helical Tm value by approximately 11 C for a central substitution.[30,31]
Both of the secondary amino acids typically found in collagen, proline and hydroxyproline, exhibited increased triple-helical thermal stability when an azGly residue was adjacently incorporated
Summary
Evaluate the effect of a central Xaa position substitution compared to CMP 1a, [H–(POG)8–NH2], the most stable natural CMP in this library, and to serve as baseline controls for the corresponding azGly-substituted CMPs. Incorporation of an azGly residue adjacent to a central, variable Yaa position in CMPs provides a reliable replacement for glycine residues.[33] The identity of the Yaa position can signi cantly affect azGly's impact on triple-helical thermal stability, ranging from high increases in the melting point of the collagen triple helix (Tm) to essentially no difference when compared to the control sequence. Our understanding of azGly's affect in collagen peptides has been limited to these speci c cases In this current work, we develop a deeper understanding by detailing the impact of neighboring Xaa residues on the ability of an azGly residue to act as a stabilizing agent (Fig. 1b). This work facilitates, advances, and informs the design of novel, ultra-stable azGly-containing CMPs for applications in a diversity of elds including biomaterials, therapeutics, peptidomimetics, and beyond
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