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

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.Integral membrane proteins (MPs) are an abundant class of proteins that play key roles in a wide range of essential cellular processes (1). To facilitate their study in vitro, detergent molecules are commonly used to extract MPs out of their native lipid–bilayer environment (2). However, the use of detergents has some inherent disadvantages. Most importantly, even though there are promising developments to improve their properties (3, 4), the insufficient mimicking of a lipid bilayer by detergent micelles often leads to destabilization and rapid loss of function of the incorporated protein (5). For many functional and structural studies, it is thus necessary to reconstitute the MP into a more stabilizing environment; for example, by replacing the detergent with amphipathic polymers (amphipols) (6) or incorporating the MP into lipid nanodiscs with a surrounding protein scaffold (7). Both approaches have proven to be valuable tools for the study of structural and functional properties of MPs (8, 9); however, a limitation remains, as transfer of MPs into any of these systems requires initial solubilization by detergent.Recently, a detergent-free approach has been described using amphipathic styrene-maleic acid copolymers (SMAs) as an alternative to solubilize MPs directly from biological membranes in the form of nanodiscs, referred to as “Lipodisq” or SMA lipid particles (10–13) (Fig. 1). The mechanism of action of SMA differs fundamentally from that of detergents: instead of disrupting the lipid bilayer completely, SMA spontaneously self-inserts and extracts intact membrane patches in the form of discoidal particles that are stabilized by a SMA annulus (14, 15). Because these nanodiscs conserve a spatially delimited native biomembrane including MPs, we term them “native nanodiscs.” One of the main advantages of this system is the straightforward extraction protocol without the need for detergent. It has been shown that the SMA polymer is capable of directly extracting native nanodiscs containing large functional protein complexes from yeast (12), bacterial proteins involved in cell division (16) and photosynthesis (17), and several members of the ABC transporter family (13). The isolation of these proteins from a variety of different organisms suggests a general applicability of SMA solubilization for all MPs, irrespective of their expression host or native organism.Open in a separate windowFig. 1.(A) Chemical structure of SMA polymers at neutral pH. For this study, a polymer with an average SMA ratio of n:m = 2:1 was used. (B) Schematic representation of a native nanodisc containing a KcsA tetramer (blue) and native lipids (green). The outer hydrophobic surface of the lipids is shielded by SMA (orange).To further explore the potential of native nanodiscs, we used the SMA polymer to isolate an oligomeric bacterial membrane protein: the tetrameric potassium channel from Streptomyces lividans (KcsA) (18), expressed in Escherichia coli. KcsA is an ideal model protein for such studies because it is well-characterized and because reconstitution studies have shown that both its function and stability are strongly affected by lipid composition (19–21). In this work, we apply SMA to prepare and purify native nanodiscs with KcsA to compare the conformational properties and stability of the protein with those in detergent micelles. In addition, we use native nanodiscs to investigate preferential lipid–protein interactions by analyzing the composition of small patches of native membrane that are copurified with the protein. Finally, we study the functional properties of KcsA on reconstitution from native nanodiscs into a planar lipid bilayer system. Our results underscore the huge potential of SMA as a membrane-solubilizing agent, as well as the use of native nanodiscs as a membrane-mimetic system for biophysical studies on ion channels, and MPs in general.