Identification of protein domains by shotgun proteolysis

Journal of Molecular Biology
Daniel Christ, Greg Winter

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

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Citations

Nov 17, 2007·Nature Protocols·Carol M Y LeeDaniel Christ
Apr 19, 2008·Nucleic Acids Research·Michael R DysonJohn McCafferty
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
Apr 17, 2015·Protein Engineering, Design & Selection : PEDS·Mark D AllenDaniel Christ
Mar 8, 2018·Nucleic Acids Research·Morgane BooneNico Callewaert
Jul 5, 2011·Methods : a Companion to Methods in Enzymology·Hayretin YumerefendiDarren J Hart

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