Abstract
Proteases are enzymes that catalyze the hydrolytic degradation of other proteins into peptides or amino acids through the digestion of the peptide bond. Promiscuous proteases that target a wide range of proteins are distinguished from specific proteases that have a narrow range of substrates. In terms of activity, endoproteases cleave their substrates at specific residues within the target proteins, whereas exoproteases cleave from one extremity and may have processive activities. Proteases are therefore very useful tools to study proteins, notably their structure or conformation. In addition, proteases can be used to probe the topology of bacterial membrane proteins. Here, we describe limited protease accessibility assays to define inner membrane protein topology and conformational changes based on digestion profiles.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Costa TR, Felisberto-Rodrigues C, Meir A, Prevost MS, Redzej A, Trokter M, Waksman G (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13:343–359
Bleves S, Lazdunski A, Filloux A (1996) Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa. J Bacteriol 178:4297–4300
Ross JA, Plano GV (2011) A C-terminal region of Yersinia pestis YscD binds the outer membrane secretin YscC. J Bacteriol 193:2276–2289
Das A, Xie YH (1998) Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins. Mol Microbiol 27:405–414
Vincent MS, Canestrari MJ, Leone P, Stathopulos J, Ize B, Zoued A, Cambillau C, Kellenberger C, Roussel A, Cascales E (2017) Characterization of the Porphyromonas gingivalis type IX secretion trans-envelope PorKLMNP core complex. J Biol Chem 292:3252–3261
Aschtgen MS, Zoued A, Lloubès R, Journet L, Cascales E (2012) The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC. Microbiology 1:71–82
Pross E, Soussoula L, Seitl I, Lupo D, Kuhn A (2016) Membrane targeting and insertion of the C-tail protein SciP. J Mol Biol 428:4218–4227
Gentschev I, Goebel W (1992) Topological and functional studies on HlyB of Escherichia coli. Mol Gen Genet 232:40–48
Allaoui A, Woestyn S, Sluiters C, Cornelis GR (1994) YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol 176:4534–4542
Jakubowski SJ, Krishnamoorthy V, Cascales E, Christie PJ (2004) Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion system. J Mol Biol 341:961–977
Ma LS, Lin JS, Lai EM (2009) An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens. J Bacteriol 191:4316–4329
Logger L, Aschtgen MS, Guérin M, Cascales E, Durand E (2016) Molecular dissection of the interface between the Type VI secretion TssM cytoplasmic domain and the TssG baseplate component. J Mol Biol 428:4424–4437
Traxler B, Boyd D, Beckwith J (1993) The topological analysis of integral cytoplasmic membrane proteins. J Membr Biol 132:1–11
van Geest M, Lolkema JS (2000) Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev 64:13–33
Cunningham K, Lill R, Crooke E, Rice M, Moore K, Wickner W, Oliver D (1989) SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J 8:955–959
Larsen RA, Thomas MG, Postle K (1999) Protonmotive force, ExbB and ligand-bound FepA drive conformational changes in TonB. Mol Microbiol 31:1809–1824
Germon P, Ray MC, Vianney A, Lazzaroni JC (2001) Energy-dependent conformational change in the TolA protein of Escherichia coli involves its N-terminal domain, TolQ, and TolR. J Bacteriol 183:4110–4114
Cascales E, Christie PJ (2004) Agrobacterium VirB10, an ATP energy sensor required for type IV secretion. Proc Natl Acad Sci U S A 101:17228–17233
Song L, Perpich JD, Wu C, Doan T, Nowakowska Z, Potempa J, Christie PJ, Cascales E, Lamont RJ, Hu B (2022) A unique bacterial secretion machinery with multiple secretion centers. Proc Natl Acad Sci U S A 119:e2119907119
Vincent MS, Comas Hervada C, Sebban-Kreuzer C, Le Guenno H, Chabalier M, Kosta A, Guerlesquin F, Mignot T, McBride MJ, Cascales E, Doan T (2022) Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium. PLoS Biol 20:e3001443
Acknowledgments
Work in EC laboratory is supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-20-CE11-0011 and ANR-20-CE11-0017) and the Fondation Bettencourt-Schueller.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chabalier, M., Doan, T., Cascales, E. (2024). Probing Protein Topology and Conformation by Limited Proteolysis. In: Journet, L., Cascales, E. (eds) Bacterial Secretion Systems . Methods in Molecular Biology, vol 2715. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3445-5_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3445-5_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3444-8
Online ISBN: 978-1-0716-3445-5
eBook Packages: Springer Protocols