Natural and Artificial Cystine Knots for Assembly of Homo- and Heterotrimeric Collagen Models

Abstract

Native collagens are molecules that are difficult to handle because of their high tendency towards aggregation and denaturation. It was discovered early on that synthetic collagenous peptides are more amenable to conformational characterization and thus can serve as useful models for structural and functional studies. Single-stranded collagenous peptides of high propensity to self-associate into triple-helical trimers were used for this purpose as well as interchain-crosslinked homotrimers assembled on synthetic scaffolds. With the growing knowledge of the biosynthetic pathways of natural collagens and the importance of their interchain disulfide crosslinks, which stabilize the triple-helical structure, native as well as de novo designed cystine knots have gained increasing attention in the assembly of triple-stranded collagen peptides. In addition, natural sequences of collagens were incorporated in order to biophysically characterize their functional epitopes. This review is focused on the methods developed over the years, and future perspectives for the production of collagen-mimicking synthetic and recombinant triple-helical homo- and heterotrimers.

Crystal Structure of Human Collagen XVIII Trimerization Domain: A Novel Collagen Trimerization Fold

“…In vitro studies of the collagen triple helix stabilized by a specific trimerization domain is then more biologically relevant. Available methods to mimic the natural trimerization of the collagen triple helix are limited to either use of disulfide cross-links48 or an exogenous trimerization domain of bacteriophage T4 36…”

Synthesis and biological applications of collagen-model triple-helical peptides

“…3,17,78,89–91 In one representative example, the following peptide chains were constructed by solid-phase methodology: α 1, which contained either 3 or 5 Gly-Pro-Hyp repeats on the N -terminus of Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val-Gly-Cys(Acm)-Gly-Gly-OH, where Acm is acetamidomethyl; α 2, which contained 3 or 5 Gly-Pro-Hyp repeats on the N -terminus of Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu-Gly-Cys(Acm)-Cys(S t Bu)-Gly-Gly-OH; and α 1′, which contained either 3 or 5 Gly-Pro-Hyp repeats on the N -terminus of Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val-Gly-Leu-Cys(S t Bu)-Gly-Gly-OH. All three peptides were synthesized using Fmoc chemistry.…”

Crystal Structure of Human Type III Collagen Gly991–Gly1032 Cystine Knot-containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries

“…The knot is formed by three interchain disulfide bonds, and it significantly stabilizes the triple helical structure. The stability imparted by the disulfide knot has been successfully used to produce collagenous peptides that otherwise would be too unstable to study (24,25). Production of these peptides involves the C-terminal extension by the bis-cysteinyl-sequence GPCCG, followed by air or glutathione oxidation at lower temperature under slightly basic conditions.…”