A continuous assay method, such as the one that utilizes an increase in fluorescence upon hydrolysis, allows for rapid and convenient kinetic evaluation of proteases. To better understand MMP behaviors toward native substrates, a variety of fluorescence resonance energy transfer (FRET)/intramolecular fluorescence energy transfer (IFET) triple-helical substrates have been constructed to examine the collagenolytic activity of MMP family members. Results of these studies have been valuable for providing insights into (a) the relative triple-helical peptidase activities of the various collagenolytic MMPs, (b) the collagen preferences of these MMPs, and (c) the relative roles of MMP domains and specific residues in efficient collagenolysis. The present chapter provides an overview of MMP FRET triple-helical substrates and describes how to construct and utilize these substrates.
Keywords: Collagen; FRET substrates; Fluorogenic substrates; Matrix metalloproteinases; Triple-helical substrates.