A new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene–maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein–lipid and protein–protein interactions.

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.membrane-protein solubilization | styrene-maleic acid copolymer | lipid-protein interactions | nanodisc | ion channels

The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as a tool in membrane research, due to its ability to directly solubilize lipid membranes into nanodisk particles without the requirement of conventional detergents. Although many variants of SMA are commercially available, so far only SMA variants with a 2:1 and 3:1 styreneto-maleic acid ratio have been used in lipid membrane studies. It is not known how SMA composition affects the solubilization behavior of SMA. Here, we systematically investigated the effect of varying the styrene/maleic acid on the properties of SMA in solution and on its interaction with membranes. Also the effect of pH was studied, because the proton concentration in the solution will affect the charge density and thereby may modulate the properties of the polymers. Using model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipids at pH > pH agg , we found that membrane solubilization is promoted by a low charge density and by a relatively high fraction of maleic acid units in the polymer. Furthermore, it was found that a collapsed conformation of the polymer is required to ensure efficient insertion into the lipid membrane and that efficient solubilization may be improved by a more homogenous distribution of the maleic acid monomer units along the polymer chain. Altogether, the results show large differences in behavior between the SMA variants tested in the various steps of solubilization. The main conclusion is that the variant with a 2:1 styrene-to-maleic acid ratio is the most efficient membrane solubilizer in a wide pH range.

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The structure, dynamics and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane, and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes, forming nanosize discoidal proteolipid particles, also referred to as native nanodiscs. Here we show, for the first time, that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified with the 2 x 34 kDa-large bacterial cation diffusion facilitator CzcD from Cupriavidus metallidurans CH34. Keywords membrane protein; nanodiscs; styrene maleic acid; NMR spectroscopy; solid-state NMR Integral membrane proteins play essential roles in numerous cellular processes. The properties of the surrounding lipid environment are crucial to their structure, dynamics and function. However, structural and biophysical characterizations by most biophysical techniques generally require extraction and purification from the native membrane. Major concerns when working with detergent-solubilized membrane proteins are related to the fact that functionally and structurally important lipids are often stripped away, and that the properties of the detergent micelle differ significantly from the planar lipid bilayer environment. An increasing number of cases of distortions of membrane protein structures in detergents have been reported.[1,2] These known problems with detergents have triggered the development of alternative non-micellar systems, such as amphiphilic polymers (amphipols), bicelles, or nanolipoprotein particles, i.e. a lipid patch surrounded by a membrane-scaffold protein. [3][4][5] A particularly interesting recent approach is the use of the beate.bersch@ibs.fr, paul.schanda@ibs.fr. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts styrene/maleic acid co-polymer (SMA), which is able to self-insert into the lipid bilayer, and to extract membrane proteins directly from the membrane, forming nanosize disc-shaped particles. [5][6][7] SMA has been successfully used to solubilize membrane proteins from reconstituted proteoliposomes [8,9], and also from native cellular membranes, forming socalled native nanodiscs. [7,[10][11][12][13][14] The stability and activity of proteins in SMA-bounded nanodiscs have been shown to be significantly higher than in detergent micelles in several instances, and some cases were reported where the stability was even higher than in the native membrane [15]. These nanodiscs have been used in functional tests, ligand binding studies, electron-microscopy, EPR and other spectroscopic characterizations (reviewed in...