The Role of Cystine Knots in Collagen Folding and Stability, Part II. Conformational Properties of (Pro‐Hyp‐Gly)_n Model Trimers with N‐ and C‐Terminal Collagen Type III Cystine Knots

Abstract

In mature collagen type III the homotrimer is C-terminally cross-linked by an interchain cystine knot consisting of three disulfide bridges of unknown connectivity. This cystine knot with two adjacent cysteine residues on each of the three alpha chains has recently been used for the synthesis and expression of model homotrimers. To investigate the origin of correct interchain cysteine pairings, (Pro-Hyp-Gly)(n) peptides of increasing triplet number and containing the biscysteinyl sequence C- and N-terminally were synthesised. The possibilities were that this origin may be thermodynamically coupled to the formation of the collagen triple helix as happens in the oxidative folding of proteins, or it could represent a post-folding event. Only with five triplets, which is known to represent the minimum number for self-association of collagenous peptides into a triple helix, air-oxidation produces the homotrimer in good yields (70 %), the rest being intrachain oxidised monomers. Increasing the number of triplets has no effect on yield suggesting the formation of kinetically trapped intermediates, which are not reshuffled by the glutathione redox buffer. N-terminal incorporation of the cystine knot is significantly less efficient in the homotrimerisation step and also in terms of triple-helix stabilisation. Compared to an artificial C-terminal cystine knot consisting of two interchain disulfide bridges, the collagen type III cystine knot produces collagenous homotrimers of remarkably high thermostability, although the concentration-independent refolding rates are not affected by the type of disulfide bridging. Since the natural cystine knot allows ready access to homotrimeric collagenous peptides of significantly enhanced triple-helix thermostability it may well represent a promising approach for the preparation of collagen-like innovative biomaterials. Conversely, the more laborious regioselectively formed artificial cystine knot still represents the only synthetic strategy for heterotrimeric collagenous peptides.

Self-assembly of synthetic collagen triple helices

“…The N and C termini in 1 and 2 were left unprotected to mimic natural collagen. Analytical experiments were performed at pH 3 to minimize the scrambling or reduction of disulfide bonds (28).…”

Preparation and characterization of monomers to tetramers of a collagen-like domain fromStreptococcus pyogenes

“…23 It may also contribute to an increased stability for a construct, especially for shorter constructs. 24 The protein yields of these various constructs dropped with increasing size, approximately halving with each CL addition. A similar effect has also been reported for globular proteins, 25 and covered a much larger molecular weight range of up to 6000 amino acids in the largest construct.…”

“…22 This sequence motif is also found in the N-terminal propeptide of type III collagen, along with 5 intrachain disulphide bonds, contributing to a significant increase in stability, of 19 C, when compared with fully reduced and carboxymethylated protein. 29 This sequence motif has been added to synthetic peptide constructs, 24,30 leading to notable increases in stability. In one example, where the recombinant construct (GlyProPro) 10 Cys 2 was studied, 30 the inclusion of the CysCys sequence led to a T m of 82 C, which is 50 C more than (GlyProPro) 10 .…”

The Structure of the Cys-rich Terminal Domain of Hydra Minicollagen, Which Is Involved in Disulfide Networks of the Nematocyst Wall

“…nantly determined by the covalent interactions between Cys residues, kinetic intermediates must be responsible for correct disulfide pairings. A folded-precursor mechanism (28,29,36) and a quasi-stochastic mechanism (30,31) (Figs. 2 and 3) this bend and the ␥-turn bring the cysteines into close vicinity for the correct intramolecular disulfide bridging.…”

“…Interestingly in the minicollagen of corals (AF507373.1) Cys-residues frequently occur after glycines in the collagenous part of the sequence. Cysteine residues, which interlink collagen chains of the same molecule are located at the ends of a triple helix to form a stable disulfide knot (35,36) or located in interruptions of the regular Gly-X-Y repeat (37). Cys-residues in the X position of collagens are highly unusual and are most likely used to link between collagen molecules.…”