LasB FRET substrate – Fluorescent Peptides

With the rise of multidrug-resistant bacteria like P. aeruginosa, the hunt for low toxicity inhibitors is paramount. A crucial part of their virulence/life cycle is cleavage of signal peptides. Type I signal peptides have a C-terminal hydrophilic domain containing a signal peptidase cleavage site commonly found in P. aeruginosa proteins that are cleaved by type I signal petidases (SPases). P. aeruginosa LasB, a type I signal peptide, is a crucial enzyme for bacterial invasion, it degrades elastin and thus aids tissue invasion, without cleavage by a SPase the protein is inactive. This peptide is an ideal candidate for enzymatic assay work in to SPase inhibitor investigations. Here we provide the substrate LasB sequence with the EDANS-Dabcyl donor quencher pair suitable for SPase inhibitor assays with FRET microscopy analysis. When this peptide is intact, fluorescence from the fluorophore (donor) EDAN is undetectable due to the proximity of the acceptor (quencher) Dabcyl. However, upon cleavage the fluorescence of the EDANS moiety, as measurably by excitation/emission 340/490nm, can be detected due to separation from the Dabcyl quencher.