Specificity-directed design of a FRET-quenched heptapeptide for assaying thermolysin-like proteases.

Thermolysin (TL) is an industrially important zinc endopeptidase, and the prototype of the M4 family of metallopeptidases. The catalytic function of TL and its relatives is typically assessed using chromogenic or more sensitive fluorescent peptides, with the latter substrates relying on Förster resonance energy transfer (FRET). Here, we demonstrate that a FRET-quenched heptapeptide designed on the basis of the enzyme's substrate specificity (Dabcyl-FKFLGKE-EDANS) is efficiently cleaved by TL and dispase (a TL-like protease) in between the Phe3 and Leu4 residues.