A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT

Sarah E Wood; Gaurav Sinsinbar; Sushanth Gudlur; Madhavan Nallani; Che-Fan Huang; Bo Liedberg; Milan Mrksich

doi:10.1002/anie.201707535

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT

Angew Chem Int Ed Engl. 2017 Dec 22;56(52):16531-16535. doi: 10.1002/anie.201707535. Epub 2017 Oct 10.

Authors

Sarah E Wood¹, Gaurav Sinsinbar², Sushanth Gudlur², Madhavan Nallani², Che-Fan Huang¹, Bo Liedberg², Milan Mrksich¹

Affiliations

¹ Departments of Chemistry and Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA.
² Center for Biomimetic Sensor Science, School of Materials Science & Engineering, Nanyang Technological University, 50 Nanyang Drive, 637553, Singapore.

PMID: 28940795
DOI: 10.1002/anie.201707535

Abstract

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k_cat /K_m value of 6.1×10⁶ L mol^-1 s^-1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Keywords: SAMDI-MS; chemical biology; peptides; protease activity; proteomics.

Publication types

Research Support, Non-U.S. Gov't