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Article Dans Une Revue Biochemical Journal Année : 2009

Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin

Philip Bardelang
  • Fonction : Auteur
Mireille Vankemmelbeke
  • Fonction : Auteur
Ying Zhang
  • Fonction : Auteur
Hannah Jarvis
  • Fonction : Auteur
Eleni Antoniadou
  • Fonction : Auteur
Sophie Rochette
  • Fonction : Auteur
Neil R Thomas
  • Fonction : Auteur
Christopher N Penfold
  • Fonction : Auteur

Résumé

We have developed a polypeptide FRET substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at position 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue in turn at four positions of the pentaglycine target site and revealed that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.

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Dates et versions

hal-00479093 , version 1 (30-04-2010)

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Philip Bardelang, Mireille Vankemmelbeke, Ying Zhang, Hannah Jarvis, Eleni Antoniadou, et al.. Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin. Biochemical Journal, 2009, 418 (3), pp.615-624. ⟨10.1042/BJ20081765⟩. ⟨hal-00479093⟩

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