title: Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore.
abstract: The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.
authors.affiliation: Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Carsten
authors.identifier: 0000-0003-0487-3311
authors.initials: C
authors.lastname: Wloka
authors.affiliation: Department of Chemistry, University of Leuven , 3001 Leuven, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Van Meervelt
authors.affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Dewi
authors.initials: D
authors.lastname: van Gelder
authors.affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Natasha
authors.initials: N
authors.lastname: Danda
authors.affiliation: Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Nienke
authors.initials: N
authors.lastname: Jager
authors.affiliation: Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Williams
authors.affiliation: Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , 9747 AG Groningen, The Netherlands.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Maglia
mesh_terms: D001688:Biological Products; D057054:Molecular Imaging / Q000379:methods*; D058608:Nanopores; D036103:Nanotechnology / Q000379:methods*; D054353:Perforin / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D025801:Ubiquitin; D054875:Ubiquitination / Q000502:physiology
chemical_list: D001688:Biological Products; D011506:Proteins; D025801:Ubiquitin; D054353:Perforin
keywords: nanopore; nanotechnology; protein modifications; single-molecule kinetics; ubiquitin