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

Sarah E. Wood, Gaurav Sinsinbar, Sushanth Gudlur, Madhavan Nallani, Che Fan Huang, Bo Liedberg^*, Milan Mrksich

^*Corresponding author for this work

Office for Research

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

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.

Original language	English (US)
Pages (from-to)	16531-16535
Number of pages	5
Journal	Angewandte Chemie - International Edition
Volume	56
Issue number	52
DOIs	https://doi.org/10.1002/anie.201707535
State	Published - Dec 22 2017

Keywords

SAMDI-MS
chemical biology
peptides
protease activity
proteomics

ASJC Scopus subject areas

Catalysis
Chemistry(all)

Access to Document

10.1002/anie.201707535

Cite this

@article{fa6f2477e8c3422d888647096fa5cc16,

title = "A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT",

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 kcat/Km 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",

author = "Wood, {Sarah E.} and Gaurav Sinsinbar and Sushanth Gudlur and Madhavan Nallani and Huang, {Che Fan} and Bo Liedberg and Milan Mrksich",

note = "Funding Information: We gratefully acknowledge support from the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt 10/20/14. We thank Batika Saxena for the illustration in Figure 1. Funding Information: We gratefully acknowledge support from the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt 10/20/14. We thank Batika Saxena for the illustration in Figure.",

year = "2017",

month = dec,

day = "22",

doi = "10.1002/anie.201707535",

language = "English (US)",

volume = "56",

pages = "16531--16535",

journal = "Angewandte Chemie - International Edition",

issn = "1433-7851",

publisher = "John Wiley and Sons Ltd",

number = "52",

}

TY - JOUR

T1 - A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT

AU - Wood, Sarah E.

AU - Sinsinbar, Gaurav

AU - Gudlur, Sushanth

AU - Nallani, Madhavan

AU - Huang, Che Fan

AU - Liedberg, Bo

AU - Mrksich, Milan

N1 - Funding Information: We gratefully acknowledge support from the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt 10/20/14. We thank Batika Saxena for the illustration in Figure 1. Funding Information: We gratefully acknowledge support from the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt 10/20/14. We thank Batika Saxena for the illustration in Figure.

PY - 2017/12/22

Y1 - 2017/12/22

N2 - 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 kcat/Km 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.

AB - 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 kcat/Km 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.

KW - SAMDI-MS

KW - chemical biology

KW - peptides

KW - protease activity

KW - proteomics

UR - http://www.scopus.com/inward/record.url?scp=85038430854&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85038430854&partnerID=8YFLogxK

U2 - 10.1002/anie.201707535

DO - 10.1002/anie.201707535

M3 - Article

C2 - 28940795

AN - SCOPUS:85038430854

SN - 1433-7851

VL - 56

SP - 16531

EP - 16535

JO - Angewandte Chemie - International Edition

JF - Angewandte Chemie - International Edition

IS - 52

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this