Mapping the Substrate Recognition Landscapes of Metalloproteases Using Comprehensive Mutagenesis

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/KM determination 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.