Abstract
The identification of protein domains within multi-domain proteins is a persistent problem. Here, we describe an experimental method (shotgun proteolysis) based on random DNA fragmentation and protease selection of the encoded polypeptides on phage for this purpose. We applied the method to the Escherichia coli genome and identified 124 protease-resistant fragments; several were re-cloned for expression as soluble fragments in bacteria, and corresponded to autonomously folding units with folding energies similar to natural protein domains (DeltaG(u)=3.8-6.6 kcal/mol). Structural information was available for approximately half of the selected proteins, which corresponded to compact, globular and domain-sized units that had been derived from a wide range of protein superfamilies. Furthermore, boundaries of the selected fragments correlated with domain boundaries as defined by bioinformatics predictions (R2=0.82; p=0.016). However, predictions were incomplete or entirely lacking for the remaining fragments, reflecting the limited proteome coverage of current bioinformatics methods. Shotgun proteolysis therefore provides a means to identify domains and other autonomously folding units on a genome-wide scale, without any prior know...Continue Reading
References
Jun 5, 1992·Journal of Molecular Biology·E M MeieringA R Fersht
Aug 11, 1991·Nucleic Acids Research·H R HoogenboomG Winter
May 20, 1988·Science·M BetterA H Horwitz
Apr 22, 1986·Biochemistry·A FontanaM Zambonin
Dec 30, 1994·Gene·A Skerra
Jun 15, 1993·Biochemistry·R W Hartley
Aug 15, 1997·Structure·C A OrengoJ M Thornton
Dec 10, 1998·Nucleic Acids Research·A Marchler-BauerS H Bryant
Oct 20, 2000·Bioinformatics·S J WheelanS H Bryant
May 31, 2002·Acta Crystallographica. Section D, Biological Crystallography·Helen M BermanChristine Zardecki
Jan 31, 2003·Proteins·Alexei SavchenkoAled M Edwards
Jun 14, 2003·Science·Cyrus ChothiaSarah A Teichmann
Dec 19, 2003·Nucleic Acids Research·Antonina AndreevaAlexey G Murzin
Dec 19, 2003·Nucleic Acids Research·Martin MaderaJulian Gough
Jul 9, 2004·Nucleic Acids Research·Jinfeng Liu, Burkhard Rost
Citations
Nov 17, 2007·Nature Protocols·Carol M Y LeeDaniel Christ
Apr 19, 2008·Nucleic Acids Research·Michael R DysonJohn McCafferty
Mar 3, 2010·BMC Bioinformatics·Eisuke ChikayamaYutaka Kuroda
Jun 28, 2006·BMC Bioinformatics·Satoshi MiyazakiShigeyuki Yokoyama
Aug 5, 2014·Expert Opinion on Biological Therapy·Marina KovalevaCaroline Barelle
Apr 23, 2013·Current Opinion in Structural Biology·Darren J Hart, Geoffrey S Waldo
Nov 13, 2007·Drug Discovery Today·Renos SavvaPaul C Driscoll
Nov 13, 2007·Drug Discovery Today·Chrisostomos ProdromouPaul C Driscoll
Aug 24, 2007·The FEBS Journal·Jian-Wei LiuDavid Ollis
Apr 17, 2015·Protein Engineering, Design & Selection : PEDS·Mark D AllenDaniel Christ
Mar 8, 2018·Nucleic Acids Research·Morgane BooneNico Callewaert
Oct 11, 2008·Biopolymers·Teppei EbinaYutaka Kuroda
Jul 28, 2010·Biochemical Society Transactions·Michael R Dyson
Jul 5, 2011·Methods : a Companion to Methods in Enzymology·Hayretin YumerefendiDarren J Hart