title: Heterotrimeric collagen peptides as fluorogenic collagenase substrates: synthesis, conformational properties, and enzymatic digestion.
abstract: The collagenase cleavage site of collagen type I, i.e., the sequence portions 772-784 (P(4)-P(9)') and 772-785 (P(4)-P(10)') of the two alpha1-chains and the sequence portion 772-784 (P(4)-P(9)') of the alpha2-chain, were assembled in an alpha1alpha2alpha1' register by C-terminal cross-linking of these peptides with an artificial cystine knot. The triple-helical conformation 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 alpha1-chain was dansylated and the alpha1'-chain was acylated with a tryptophan residue to place in spatial proximity the two chromophores for an efficient fluorescence resonance energy transfer. Although the incorporation of the two N-terminal chromophores leads to partial destabilization of the overall triple-helical fold, the heterotrimer behaved as a collagen-like substrate of the matrix metalloproteinases MMP-1 and MMP-13. Cleavage of the fluorogenic heterotrimer leads to a 6-fold increase in fluorescence intensity, thus making it a useful fluorogenic substrate for interstitial collagenases. With this folded heterotrimeric collagen molecule it was shown that fluorescence resonance energy transfer, as applied so far only for the design of linear fluorogenic enzyme substrates, can also be exploited in conformation dependency.
authors.affiliation: Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
authors.forename: J C
authors.initials: JC
authors.lastname: Müller
authors.forename: J
authors.initials: J
authors.lastname: Ottl
authors.forename: L
authors.initials: L
authors.lastname: Moroder
mesh_terms: D000818:Animals; D001665:Binding Sites; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D003094:Collagen; D017364:Collagenases