Self-assembling nanostructures in aqueous mixtures of bilayer-forming lipids and micelle-forming surfactants are relevant to in vitro studies on biological and synthetic membranes and membrane proteins. Considerable efforts are currently underway to replace conventional detergents by milder alternatives such as styrene/maleic acid (SMA) copolymers and fluorinated surfactants. However, these compounds and their nanosized assemblies remain poorly understood as regards their interactions with lipid membranes, particularly, the thermodynamics of membrane partitioning and solubilisation. Using (19)F and (31)P nuclear magnetic resonance spectroscopy, static and dynamic light scattering, and isothermal titration calorimetry, we have systematically investigated the aggregational state of a zwitterionic bilayer-forming phospholipid upon exposure to an SMA polymer with a styrene/maleic acid ratio of 3 : 1 or to a fluorinated octyl phosphocholine derivative called F(6)OPC. The lipid interactions of SMA(3 : 1) and F(6)OPC can be thermodynamically conceptualised within the framework of a three-stage model that treats bilayer vesicles, discoidal or micellar nanostructures, and the aqueous solution as distinct pseudophases. The exceptional solubilising power of SMA(3 : 1) is reflected in very low membrane-saturating and solubilising polymer/lipid molar ratios of 0.10 and 0.15, respectively. Although F(6)OPC saturates bilayers at an even lower molar ratio of 0.031, this nondetergent does not solubilise lipids even at >1000-fold molar excess, thus highlighting fundamental differences between these two types of mild membrane-mimetic systems. We rationalise these findings in terms of a new classification of surfactants based on bilayer-to-micelle transfer free energies and discuss practical implications for membrane-protein research.

The dynamic ribosome–translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3–7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.

Copolymers of styrene and maleic acid (SMA) have gained great attention as alternatives to conventional detergents, as they offer decisive advantages for studying membrane proteins and lipids in vitro. These polymers self-insert into artificial and biological membranes and, at sufficiently high concentrations, solubilise them into disc-shaped nanostructures containing a lipid bilayer core surrounded by a polymer belt. We have used (31)P nuclear magnetic resonance spectroscopy and dynamic light scattering to systematically study the solubilisation of vesicles composed of saturated or unsaturated phospholipids by an SMA copolymer with a 3 : 1 styrene/maleic acid molar ratio at different temperatures. Solubilisation was thermodynamically rationalised in terms of a three-stage model that treats various lipid/polymer aggregates as pseudophases. The solubilising capacity of SMA(3 : 1) towards a saturated lipid is higher in the gel than in the liquid-crystalline state of the membrane even though solubilisation is slower. Although the solubilisation of mixed fluid membranes is non-selective, the presence of a non-bilayer phospholipid lowers the threshold at which the membrane becomes saturated with SMA(3 : 1) but raises the polymer concentration required for complete solubilisation. Both of these trends can be explained by considering the vesicle-to-nanodisc transfer free energies of the lipid and the polymer. On the basis of the phase diagrams thus obtained, re-association of polymer-solubilised lipids with vesicles is possible under mild conditions, which has implications for the reconstitution of proteins and lipids from nanodiscs into vesicular membranes. Finally, the phase diagrams provide evidence for the absence of free SMA(3 : 1) in vesicular lipid suspensions.

Some styrene/maleic acid (SMA) copolymers solubilise membrane lipids and proteins to form polymer-bounded nanodiscs termed SMA/lipid particles (SMALPs). Although SMALPs preserve a lipid-bilayer core, they appear to be more dynamic than other membrane mimics. We used time-resolved Förster resonance energy transfer and small-angle neutron scattering to determine the kinetics and the mechanisms of phospholipid transfer among SMALPs. In contrast with vesicles or protein-bounded nanodiscs, SMALPs exchange lipids not only by monomer diffusion but also by fast collisional transfer. Under typical experimental conditions, lipid exchange occurs within seconds in the case of SMALPs but takes minutes to days in the other bilayer particles. The diffusional and second-order collisional exchange rate constants for SMALPs at 30 °C are kdif = 0.287 s−1 and kcol = 222 M−1s−1, respectively. Together with the fast kinetics, the observed invariability of the rate constants with probe hydrophobicity and the moderate activation enthalpy of ~70 kJ mol−1 imply that lipids exchange through a “hydrocarbon continuum” enabled by the flexible nature of the SMA belt surrounding the lipid-bilayer core. Owing to their fast lipid-exchange kinetics, SMALPs represent highly dynamic equilibrium rather than kinetically trapped membrane mimics, which has important implications for studying protein/lipid interactions in polymer-bounded nanodiscs.