Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries

Biochemistry
N DekkerM R Egmond

Abstract

Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that specifically cleaves between two consecutive basic residues. In this study we have investigated the substrate specificity of OmpT using spatially addressed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg/Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. The peptide contained the aminobenzoyl (Abz) fluorophore, which was internally quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membrane with the small, water-soluble protease trypsin resulted in highly fluorescent peptide SPOTs. However, no peptide cleavage was observed after incubation with detergent-solubilized OmpT, a macromolecular complex with an estimated molecular mass of 180 kDa. This problem could be solved by the introduction of a long, polar polyoxyethylene glycol linker between the membrane support and the peptide. Peptide libraries for the P(2), P(1), P(1)', and P(2)' positions in the substrate were screened with OmpT, and peptides of positive SPOTs were resynthesized and subjected to kinetic measurements in solution. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover number k(cat) of 40 s(-)(1), which is 12-fold higher than t...Continue Reading

References

Apr 20, 1967·Biochemical and Biophysical Research Communications·I Schechter, A Berger
Apr 12, 1994·Proceedings of the National Academy of Sciences of the United States of America·M MeldalF I Auzanneau
Feb 1, 1994·Analytical Biochemistry·Y Duan, R A Laursen
Jan 1, 1994·Journal of Bacteriology·A KaufmannU Henning
Oct 15, 1995·Structure·E Pebay-PeyroulaP A Timmins
Apr 1, 1997·The Journal of Peptide Research : Official Journal of the American Peptide Society·W Tegge, R Frank

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Citations

Jan 6, 2012·Applied Microbiology and Biotechnology·Jerica Sabotič, Janko Kos
Dec 4, 2009·Biotechnology Letters·Wenli HuangXiaojun Zhao
May 24, 2011·Molecular Biotechnology·Alexandra ThieleMike Schutkowski
Sep 27, 2002·Current Opinion in Biotechnology·Ulf ReimerJens Schneider-Mergener
Jan 28, 2003·Current Opinion in Chemical Biology·Benjamin E Turk, Lewis C Cantley
Jun 19, 2012·Nature Methods·Cong WuNeil L Kelleher
Jul 20, 2007·Molecular BioSystems·Dehua Pei, Anne-Sophie Wavreille
Mar 16, 2002·European Journal of Biochemistry·R Arjen KramerMaarten R Egmond
Nov 8, 2008·Journal of Biochemistry·Denis DuchéRoland Lloubès
Aug 26, 2004·Antimicrobial Agents and Chemotherapy·Linda TomasinsigMargherita Zanetti
Feb 6, 2007·Journal of Bacteriology·Muriel MasiRajeev Misra
Nov 7, 2006·Journal of Bacteriology·Bum-Yeol HwangGeorge Georgiou
Aug 20, 2004·Journal of Bacteriology·John D McCarterGeorge Georgiou
Feb 12, 2011·BMC Evolutionary Biology·Johanna HaikoTimo K Korhonen
May 4, 2005·Proceedings of the National Academy of Sciences of the United States of America·Navin VaradarajanBrent L Iverson
Feb 28, 2002·Bioscience, Biotechnology, and Biochemistry·Kazuaki OkunoShinichi Kinoshita
Mar 9, 2004·Methods : a Companion to Methods in Enzymology·Benjamin E Turk, Lewis C Cantley
May 14, 2009·Chembiochem : a European Journal of Chemical Biology·Rudolf Volkmer
Sep 4, 2009·Journal of Cellular and Molecular Medicine·Thomas H Yun, James H Morrissey
Apr 28, 2006·Chembiochem : a European Journal of Chemical Biology·Javier F Chaparro-RiggersUwe Bornscheuer
Aug 14, 2010·Journal of Thrombosis and Haemostasis : JTH·M Valls SerónJ C M Meijers
Sep 11, 2007·Biophysical Journal·Marilisa NeriPaolo Carloni
Aug 6, 2004·International Journal of Medical Microbiology : IJMM·Maini Kukkonen, Timo K Korhonen
Aug 19, 2004·Biophysical Journal·Marc Baaden, Mark S P Sansom
Apr 1, 2015·The FEBS Journal·Liana Tsiatsiani, Albert J R Heck
Mar 12, 2004·Biochemical and Biophysical Research Communications·Ryuji KatoHiroyuki Honda
Apr 1, 2015·Infection and Immunity·John R BrannonHervé Le Moual
Sep 10, 2015·Journal of Bacteriology·John R BrannonHervé Le Moual
Apr 25, 2007·Molecular Membrane Biology·Yihfen T YenChristos Stathopoulos
Aug 22, 2007·Molecular Membrane Biology·Victoria Hritonenko, Christos Stathopoulos

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