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A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site ( P4- P′ 910) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp) 5triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple-stranded cystine peptide. While the single chains showed no tendency to self-association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature ( Tm) of 33°C. This triple-helical collagen-like peptide is cleaved by the full-length human neutrophil collagenase (MMP-8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe 79-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits Kmvalues of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP-8 for the triple-helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site () of type I collagen linked to a C‐terminal cystine‐knot, and N‐terminally extended with (Gly‐Pro‐Hyp)5triplets for stabilization of the triple‐helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple‐stranded cystine peptide. While the single chains showed no tendency to self‐association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen‐like CD spectrum at room temperature and a melting temperature (Tm) of 33°C. This triple‐helical collagen‐like peptide is cleaved by the full‐length human neutrophil collagenase (MMP‐8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe79‐MMP‐8 domain of HNC. Moreover, the full‐length enzyme exhibits Kmvalues of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP‐8 for the triple‐helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.