title: Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs.
abstract: A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.
authors.affiliation: Membrane Biochemistry and Biophysics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands; j.m.dorr@uu.nl.
authors.forename: Jonas M
authors.initials: JM
authors.lastname: Dörr
authors.affiliation: Membrane Biochemistry and Biophysics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands;
authors.forename: Martijn C
authors.initials: MC
authors.lastname: Koorengevel
authors.affiliation: Membrane Biochemistry and Biophysics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands;
authors.forename: Marre
authors.initials: M
authors.lastname: Schäfer
authors.affiliation: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands; and.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Prokofyev
authors.affiliation: Membrane Biochemistry and Biophysics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands;
authors.forename: Stefan
authors.initials: S
authors.lastname: Scheidelaar
authors.affiliation: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands; and.
authors.forename: Elwin A W
authors.initials: EA
authors.lastname: van der Cruijsen
authors.affiliation: School of Bio Sciences, University of Birmingham, Edgbaston Birmingham B15 2TT, United Kingdom.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Dafforn
authors.affiliation: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands; and.
authors.forename: Marc
authors.initials: M
authors.lastname: Baldus
authors.affiliation: Membrane Biochemistry and Biophysics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands;
authors.forename: J Antoinette
authors.initials: JA
authors.lastname: Killian
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002308:Cardiolipins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D010715:Phosphatidylglycerols / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D048170:Streptomyces lividans / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002308:Cardiolipins; D008051:Lipid Bilayers; D010715:Phosphatidylglycerols; D015221:Potassium Channels; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel
keywords: ion channels; lipid–protein interactions; membrane–protein solubilization; nanodisc; styrene-maleic acid copolymer