Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K⁺ channel: The power of native nanodiscs

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-12, Vol.111 (52), p.18607-18612
• Main Authors: Dörr, Jonas M., Koorengevel, Martijn C., Schäfer, Marre, Prokofyev, Alexander V., Scheidelaar, Stefan, van der Cruijsen, Elwin A. W., Dafforn, Timothy R., Baldus, Marc, Killian, J. Antoinette
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Biochemistry; Biological Sciences; Cardiolipins - chemistry; Chromatography; Detergents; Escherichia coli; Escherichia coli - genetics; Experiments; Fluorescence; Lipid bilayers; Lipid Bilayers - chemistry; Lipids; Membrane proteins; Membranes; Micelles; Nanostructures - chemistry; Phosphatidylglycerols - chemistry; Polymers; Potassium channels; Potassium Channels - chemistry; Potassium Channels - genetics; Potassium Channels - isolation & purification; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Solubilization; Streptomyces lividans - chemistry; Streptomyces lividans - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1416205112

Significance The study of membrane proteins is often hampered by their tendency to misfold when extracted by detergent. Here, we explore a detergent-free approach to isolating membrane proteins while retaining their native lipid environment, making use of an amphipathic polymer that solubilizes intact membrane patches in the form of nanodiscs. Using a potassium channel as a model protein, we show that these “native nanodiscs” are highly thermostable particles that are suitable for spectroscopic studies, allowing structural characterization of the protein in its native environment and direct analysis of the lipids in its immediate surroundings. We also demonstrate that the channel can be reconstituted from nanodiscs into planar lipid bilayers for functional characterization, thus making native nanodiscs an excellent alternative to detergent solubilization. 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.