title: Determining the Substrate Specificity of Matrix Metalloproteases using Fluorogenic Peptide Substrates.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, Jupiter, FL, 33458, USA.
authors.forename: Maciej J
authors.initials: MJ
authors.lastname: Stawikowski
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, Jupiter, FL, 33458, USA.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Knapinska
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, Jupiter, FL, 33458, USA. fieldsg@fau.edu.|Department of Chemistry, The Scripps Research Institute/Scripps Florida, Jupiter, FL, 33458, USA. fieldsg@fau.edu.|Departments of Chemistry and Biology, Torrey Pines Institute for Molecular Studies, 11350 Village Pkwy, Port St. Lucie, FL, 34987, USA. fieldsg@fau.edu.
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D020782:Matrix Metalloproteinases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D000072417:Protein Domains; D013379:Substrate Specificity
chemical_list: D003094:Collagen; D020782:Matrix Metalloproteinases
keywords: Collagen; FRET substrates; Fluorogenic substrates; Matrix metalloproteinases; Triple-helical substrates