title: Mapping the Substrate Recognition Landscapes of Metalloproteases Using Comprehensive Mutagenesis.
abstract: Protease specificity is controlled by exosites, which capture and orient the substrate, and the active site, which binds substrate residues near the P1-P1' scissile bond and catalyzes peptide hydrolysis. Techniques used to identify critical contact points between a protease and its substrate can be time consuming and labor-intensive. Screening tools such as phage display have been revitalized with the emergence of high-throughput sequencing technology, and can be used to interrogate protease substrate specificity. This article will outline a method for creating and screening a comprehensive mutagenesis substrate phage display library. High-throughput sequencing of uncleaved phage at various reaction time points enables kcat/KMdetermination for every possible single amino acid substitution at each position of the substrate, providing unprecedented resolution for the interaction between a protease and its substrate.
authors.affiliation: Thrombosis and Atherosclerosis Research Institute and Department of Medicine, McMaster University, Room C5-117, 237 Barton St. East, Hamilton, ON, Canada, L8L 2X2. colin.kretz@taari.ca.
authors.forename: Colin A
authors.initials: CA
authors.lastname: Kretz
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D059014:High-Throughput Nucleotide Sequencing; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D019151:Peptide Library; D045726:Metalloproteases
keywords: High-throughput sequencing; Metalloprotease; Mutagenesis; Phage display; Protease; Substrate