Heterotrimeric collagen peptides as fluorogenic collagenase substrates: Synthesis, conformational properties, and enzymatic digestion

The collagenase cleavage site of collagen type I, i.e., the sequence portio ns 772-784 (P-4-P-9') and 772-785 (P-4-P-10') Of the two alpha 1-chains and the sequence portion 772-784 (P-4-P-9') Of the alpha 2-chain, were assembl ed in an alpha 1 alpha 2 alpha 1' register by C-terminal cross-linking of t hese peptides with an artificial cystine knot. The triple-helical conformat ion of the construct was stabilized by N-terminal extensions with (Gly-Pro- Hyp)(5) repeats. The gaps in the sequence alignment were filled up, and the alpha 1-chain was dansylated and the alpha 1'-chain was acylated with a tr yptophan residue to place in spatial proximity the two chromophores for an efficient fluorescence resonance energy transfer. Although the incorporatio n of the two N-terminal chromophores leads to partial destabilization of th e overall triple-helical fold, the heterotrimer behaved as a collagen-like substrate of the matrix metalloproteinases MMP-1 and MMP-13. Cleavage of th e fluorogenic heterotrimer leads to a 6-fold increase in fluorescence inten sity, thus making it a useful fluorogenic substrate for interstitial collag enases. With this folded heterotrimeric collagen molecule it was shown that fluorescence resonance energy transfer, as applied so far only for the des ign of linear fluorogenic enzyme substrates, can also be exploited in confo rmation dependency.