## 3 Amphipathic Polymers and Nanodisc Formation  
A growing family of SMA copolymers are being used to separate membranes into native nanodiscs that retain the original protein:lipid complexes found in cells. These polymers contain sequences of styrene (S) and maleic acid (MA) residues or their derivatives and are activated by hydrolysis of styrene maleimide (Figure 2). The most commonly used ratios of S:MA residues are between 2:1 and 3:1, which provides an optimal balance of hydrophobicity and polarity to allow insertion into bilayers while maintaining solubility and membrane fragmentation activity. The negatively charged MA sidechains provide solubility to the polymers and nanodiscs across the physiological pH range. Addition of the polymer at a critical concentration of around 1% to whole cells or membranes spontaneously yields water soluble nanodiscs that are stable and can be reconstituted after freeze-drying and storage while retaining protein activity [14]. A variety of SMA reagents are available commercially and more are in development. Polyscope (Polyscope, Geleen, Netherlands) offers XIRAN(r)25010 and 30010 reagents, which have S:MA ratios of 3:1 and 2.3:1, respectively. Hydrolyzed SMA2000 and SMA3000 copolymers from Cray Valley (USA) contain S:MA ratios of 2:1 and 3:1, respectively, and are similar to Lipodisq(r)reagents. Thus far, the SMA2000 and the XIRAN reagents have been widely used for solubilizing bacterial, fungal, plant, and mammalian membrane proteins that contain anywhere between monomeric and multimeric proteins with 1 to 48 transmembrane \(\alpha\)-helices [15; 16; 17]. The nanodiscs formed spontaneously by adding activated SMA solutions have diameters of around 10 nm, although they can range from 6 to 30 nm depending on the protocol and SMA variant. For example, SMA(3:1) polymer solubilizes liposomes made of synthetic lipids and transfers their contents into 6-10 nm nanodiscs [18]. A fuzzy annulus of polydispersed polymers surrounds an inner patch of bilayer, as seen by small angle neutron scattering (SANS) experiments using hydrogenated and deuterated lipids [19]. The sytrenes interact with the acyl tails of lipids as can be evidenced by intermolecular contacts observed using NMR [19], although the length and sequence polydispersity of current polymers as well as their non-specific interactions with memteins generally limit the experimental resolution. The mechanism by which SMA forms nanodiscs involves at least two stages (Figure 1). The copolymer attaches to the membrane by nonspecific insertion of hydrophobic styrene rings between the lipid acyl tails while preserving conformations or the protein and directly associated lipid [20]. Above0.a critical concentration, the accumulated copolymer destabilizes and perforates the bilayer, which fragments to form nanodiscs surrounded by a boundary of multiple polymer molecules. The molecular processes involved can be modelled by molecular dynamics simulations, as illustrated by trajectories of SMA(2:1) and SMA(3:1) copolymers as they bind and perforate dimyristoylphosphatidylcholine (DMPC) bilayer and extract lipid patches into nanodiscs [21]. Unlike classical detergs, SMA polymers have very mild solubilizing activity and hence induce minimal alteration of memteins [22; 23], allowing the position of protein-bound lipids to be kept intact including the packing of lipid acyl chains [19; 24]. The optimal pH range at which SMA copolymers are effective at solubilizing membranes is between 7 and 9. Low salt levels can increase polymer solubility further [25], although higher salt may be necessary in the presence of millimolar divalent cations. Such cations otherwise bind the negatively charged MA groups and could cause polymer precipitation. A ratio of 2:1 for S:MA ratios appears most effective at solubilizing as this perturbs the lipid bilayer least [19], although other polymer types will behave differently.