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 α 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.