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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year

IMPACT FACTOR 2016: 3.273

CiteScore 2016: 3.01

SCImago Journal Rank (SJR) 2016: 1.679
Source Normalized Impact per Paper (SNIP) 2016: 0.800

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Volume 393, Issue 9 (Sep 2012)


Identifi cation of protease exosite-interacting peptides that enhance substrate cleavage kinetics

Abeer M. Jabaiah / Jennifer A. Getz / Witold A. Witkowski
  • Department of Chemistry, University of Massachusetts, Amherst, 710 North Pleasant Street, Amherst, MA 01003-9336, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Jeanne A. Hardy
  • Department of Chemistry, University of Massachusetts, Amherst, 710 North Pleasant Street, Amherst, MA 01003-9336, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Patrick S. Daugherty


Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specifi city and catalytic effi ciency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modifi ed the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color fl ow cytometry. Exosite CLiPS (eCLiPS) identifi ed exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, proteaseactivated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profi le the ligand specifi city of protease exosites and to develop improved substrates.

This article offers supplementary material which is provided at the end of the article.

Keywords: CLiPS; exosite; protease; substrate; thrombin

About the article

Corresponding author

Received: 2012-03-20

Accepted: 2012-05-06

Published in Print: 2012-09-01

Citation Information: , ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2012-0162.

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©2012 by Walter de Gruyter Berlin Boston. Copyright Clearance Center

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