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Styrene-maleic acid (SMA) copolymers can extract membrane proteins from native membranes along with lipids as nanodiscs. Preparation with SMA is fast, cost-effective, and captures the native protein-lipid interactions. On the other hand, cryo-EM has become increasingly successful and efficient for structural determinations of membrane proteins, with biochemical sample preparation often the bottleneck. Three recent cryo-EM studies on the efflux transporter AcrB and the alternative complex III: cyt c oxidase supercomplex have demonstrated the potential of SMA nanodisc samples to yield high-resolution structure information of membrane proteins.

Most membrane-solubilising amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) carry high negative charge densities. Their polyanionic character results in strong Coulombic repulsion, both between polymer chains and lipid membranes during the solubilisation process as well as among polymer-encapsulated nanodiscs after solubilisation. Coulombic repulsion is attenuated by charge screening and, more efficiently, by counterion association, which is particularly strong for multivalent cations binding to polyanionic copolymers. Here, we investigated the effects of the two common alkaline earth metal ions Mg2+ and Ca2+ on the solubilisation properties of and the nanodiscs formed by DIBMA. By quantifying the kinetics and the equilibrium efficiency of lipid solubilisation by static and dynamic light scattering, respectively, we found that millimolar concentrations of Mg2+ or Ca2+ accelerated DIBMA-mediated lipid solubilisation several-fold and resulted in considerably smaller nanodiscs than without divalent cations. Time-resolved Forster resonance energy transfer spectroscopy revealed that collisional transfer of phospholipids among nanodiscs was up to ∼20 and ∼25 times faster in the presence of 10 mM Mg2+ or 7.5 mM Ca2+ than in the absence of divalent cations. These major effects of Mg2+ and Ca2+ contrasted with a moderate influence on the morphology and the thermotropic phase behaviour of the nanodiscs. Finally, we compared the yields of membrane-protein extraction from Escherichia coli membranes, which increased by up to two-fold upon addition of Mg2+ or Ca2+. None of these effects could be explained by Coulombic screening alone, as the change in ionic strength resulting from low millimolar concentrations of divalent cations was minor. Thus, we conclude that Mg2+ and Ca2+ specifically associated with DIBMA to neutralise part of the polymer's carboxylate groups.

The internal profile across the bilayer reveals important structural information regarding the crystallinity of acyl chains or the positions of encapsulated species. Here, we demonstrate that a simple five-layer-core-shell discoidal model can be employed to best fit the extended-q small angle X-ray scattering (SAXS) data and resolve the bilayer internal structure (with sub-nanometer resolution) of a nanoscale discoidal system comprised of a mixture of long- and short- chain lipids (known as “bicelles”). In contrast to the traditional core-shell discoidal model, the detailed structure in the hydrophobic core such as the methylene and methyl groups can be distinguished via this model. The refined model is validated by the SAXS data of bicelles whose electron scattering length density of the hydrophobic core is adjusted by the addition of a long-chain lipid with a fluorine-end group. The higher resolution of the bilayer internal structure can be employed to advance our understanding of the interaction and conformation of the membrane and associated molecules, such as membrane-associated proteins and locations of entrapped species in the lipid nanoparticles.

Hydrogen deuterium exchange mass spectrometry (H/DX MS) provides a quantitative comparison of the relative rates of exchange of amide protons for solvent deuterons. In turn, the rate of amide exchange depends on a complex combination of the stability of local secondary structure, solvent accessibility, and dynamics. H/DX MS has, therefore, been widely used to probe structure and function of soluble proteins, but its application to membrane proteins was limited previously to detergent solubilized samples. The large excess of lipids from model membranes, or from membrane fractions derived from in vivo samples, presents challenges with mass spectrometry. The lipid nanodisc platform, consisting of apolipoprotein A-derived membrane scaffold proteins, provides a native like membrane environment in which to capture analyte membrane proteins with a well defined, and low, ratio of lipid to protein. Membrane proteins in lipid nanodiscs are amenable to H/DX MS, and this is expected to lead to a rapid increase in the number of membrane proteins subjected to this analysis. Here we review the few literature examples of the application of H/DX MS to membrane proteins in nanodiscs. The incremental improvements in the experimental workflow of the H/DX MS are described and potential applications of this approach to study membrane proteins are described.

Understanding how stereochemistry affects interactions with cell membranes is important for effective drug development. Chirality has been shown to greatly effect pharmaceutical distribution and metabolism within the cell. However it has been thought that interactions with, and passive diffusion through, the membrane are not stereochemically selective. Various studies have produced conflicting results regarding whether interactions with lipid bilayers are or can be stereoselective. In the current work, stereoselective interactions between a pair of atropisomers, R-(+)/(S)-(-) 1,1'-Bi-2-naphthol, and sphingomyelin nanodisc bilayers, are demonstrated. This is accomplished using nanodisc electrokinetic chromatography, demonstrating that this approach is sensitive to subtle differences in affinity between small molecule probes and lipid bilayers. Using the same approach, no evidence of stereoselectivity was observed using enantiomer or diastereomer probes of varied chemistry and structure.

Styrene-maleic acid copolymers (SMA) have been gaining interest in the field of membrane research due to their ability to solubilize membranes into nanodics. The SMA molecules act as an amphipathic belt that surrounds the nanodiscs, whereby the hydrophobic styrene moieties can insert in between the lipid acyl chains. Here we used SMA variants with different styrene-to-maleic acid ratio (i.e. 2:1, 3:1 and 4:1) to investigate how lipid packing in the nanodiscs is affected by the presence of the polymers and how it depends on polymer composition. This was done by analyzing the thermotropic properties of a series of saturated phosphatidylcholines in nanodiscs using laurdan fluorescence and differential scanning calorimetry. In all cases it was found that the temperature of the main phase transition (Tm) of the lipids in the nanodiscs is downshifted and that its cooperativity is strongly reduced as compared to the situation in vesicles. These effects were least pronounced for lipids in nanodiscs bounded by SMA 2:1. Unexpected trends were observed for the calorimetric enthalpy of the transition, suggesting that the polymer itself contributes, possibly by rearranging around the nanodiscs when the lipids adopt the fluid phase. Finally, distinct differences in morphology were observed for nanodiscs at relatively high polymer concentrations, depending on the SMA variant used. Overall, the results suggest that the extent of preservation of native thermodynamic properties of the lipids as well as the stability of the nanodiscs at high polymer concentrations is better for SMA 2:1 than for the other SMA variants.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key player in regulating the process of excytosis, including insulin secretion. Granuphilin, a tandem C2 domain containing protein, mediates the docking of insulin granules onto plasma membrane. The C2A domain plays key roles in this process through interaction with PI(4,5)P2. In this study, we have investigated the molecular recognition mechanism of granuphilin-C2A domain to PI(4,5)P2 head group, and further to PI(4,5)P2-nanodisc by NMR, ITC, MST and SEC methods. Our results demonstrate that PI(4,5)P2 binds to the concave surface of granuphilin-C2A domain. The key residues involved in the binding were validated by mutation analysis.