Styrene/maleic acid copolymers (SMA) have recently attracted great interest for in vitro studies of membrane proteins, as they self-insert into and fragment biological membranes to form polymer-bounded nanodiscs that provide a native-like lipid-bilayer environment. SMA copolymers are available in different styrene/maleic acid ratios and chain lengths and, thus, possess different charge densities, hydrophobicities, and solubilisation properties. Here, we studied the equilibrium solubilisation properties of the most commonly used copolymer, SMA(2:1), by monitoring the formation of nanodiscs from phospholipid vesicles using 31P nuclear magnetic resonance spectroscopy, dynamic light scattering, and differential scanning calorimetry. Comparison of SMA(2:1) phase diagrams with those of SMA(3:1) and diisobutylene/maleic acid (DIBMA) revealed that, on a mass concentration scale, SMA(2:1) is the most efficient membrane solubiliser, despite its relatively mild effects on the thermotropic phase behaviour of solubilised lipids. In contrast with previous kinetic studies, our equilibrium experiments demonstrate that the solubilisation of phospholipid bilayers by SMA(2:1) is most efficient at moderately alkaline pH values. This pH dependence was also observed for the solubilisation of native Escherichia coli membranes, for which SMA(2:1) again turned out to be the most powerful solubiliser in terms of the total amounts of membrane proteins extracted.

Membrane proteins can be examined in near‐native lipid‐bilayer environments with the advent of polymer‐encapsulated nanodiscs. These nanodiscs self‐assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent‐solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral—yet water‐soluble—polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo‐DIBMA and Sulfo‐SMA(2:1). Sulfo‐DIBMA and Sulfo‐SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein–lipid interactions through lab‐on‐a‐chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

Styrene/maleic acid (SMA) and related copolymers are attracting great interest because they solubilise membrane proteins and lipids to form polymer-encapsulated nanodiscs. These nanodiscs retain a lipid-bilayer core surrounded by a polymer rim and can harbour a membrane protein or a membrane-protein complex. SMA exists in different styrene/maleic acid molar ratios, which results in differences in hydrophobicity and solubilisation properties. We have recently demonstrated fast collisional lipid transfer among nanodiscs encapsulated by the relatively hydrophobic copolymer SMA(3:1). Here, we used time-resolved Förster resonance energy transfer to quantify the lipid-transfer kinetics among nanodiscs bounded by SMA(2:1), a less hydrophobic copolymer that is superior in terms of lipid and membrane-protein solubilisation. Moreover, we assessed the effects of ionic strength and, thereby, the role of Coulombic repulsion in the exchange of lipid molecules among these polyanionic nanodiscs. Collisional lipid transfer was slower among SMA(2:1) nanodiscs (k = 5.9 M s) than among SMA(3:1) nanodiscs (k = 222 M s) but still two to three orders of magnitude faster than diffusional lipid exchange among protein-encapsulated nanodiscs or vesicles. Increasing ionic strength accelerated lipid transfer in a manner predicted by the Davies equation, an empirical extension of the Debye-Hückel limiting law, or an extended equation taking into account the finite size of the nanodiscs. Using the latter approach, quantitative agreement between experiment and theory was achieved for an effective nanodisc charge number of z ≈ -33, which is an order of magnitude less than their nominal overall charge.

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle‐forming detergents are particularly useful for in vitro membrane‐protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane‐protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant‐bearing terphenyl unit as a hydrophobic group, designated terphenyl‐cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self‐assembly behavior, as well as detergent efficacy for membrane‐protein stabilization. TPM‐Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM‐Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n‐dodecyl‐β‐d‐maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM‐L3 was particularly effective at stabilizing the human β2 adrenergic receptor (β2AR), a G‐protein coupled receptor, and its complex with Gs protein. Thus, the current study not only provides novel detergent tools that are useful for membrane‐protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.

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