Mechanism of gating and partial agonist action in the glycine receptor.

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.

Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

Cellular Prion Protein Conformational Shift after Liquid-Liquid Phase Separation Regulated by a Polymeric Antagonist and Mutations.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins has been associated with neurodegenerative diseases, although direct mechanisms are poorly defined. Here, we report on a maturation process for the cellular prion protein (PrPC) that involves a conformational change after LLPS and is regulated by mutations and poly(4-styrenesulfonic acid-co-maleic acid) (PSCMA), a molecule that has been reported to rescue Alzheimer's disease-related cognitive deficits by antagonizing the interaction between PrPC and amyloid-ß oligomers (Aßo). We show that PSCMA can induce reentrant LLPS of PrPC and lower the saturation concentration (Csat) of PrPC by 100-fold. Regardless of the induction method, PrPC molecules subsequently undergo a maturation process to restrict molecular motion in a more solid-like state. The PSCMA-induced LLPS of PrPC stabilizes the intermediate LLPS conformational state detected by NMR, though the final matured ß-sheet-rich state of PrPC is indistinguishable between induction conditions. The disease-associated E200 K mutation of PrPC also accelerates maturation. This post-LLPS shift in protein conformation and dynamics is a possible mechanism of LLPS-induced neurodegeneration.

Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

In Situ Eutectic Formation in a Polymeric Matrix via Hot-Melt Reactive Extrusion and the Use of Partial Least Squares Regression Modeling for Reaction Yield Determination.

There has been a significant volume of work investigating the design and synthesis of new crystalline multicomponent systems via examining complementary functional groups that can reliably interact through the formation of noncovalent bonds, such as hydrogen bonds (H-bonds). Crystalline multicomponent molecular adducts formed using this approach, such as cocrystals, salts, and eutectics, have emerged as drug product intermediates that can lead to effective drug property modifications. Recent advancement in the production for these multicomponent molecular adducts has moved from batch techniques that rely upon intensive solvent use to those that are solvent-free, continuous, and industry-ready, such as reactive extrusion. In this study, a novel eutectic system was found when processing albendazole and maleic acid at a 1:2 molar ratio and successfully prepared using mechanochemical methods including liquid-assisted grinding and hot-melt reactive extrusion. The produced eutectic was characterized to exhibit a 100 °C reduction in melting temperature and enhanced dissolution performance (>12-fold increase at 2 h point), when compared to the native drug compound. To remove handling of the eutectic as a formulation intermediate, an end-to-end continuous-manufacturing-ready process enables feeding of the raw parent reagents in their respective natural forms along with a chosen polymeric excipient, Eudragit EPO. The formation of the eutectic was confirmed to have taken place in situ in the presence of the polymer, with the reaction yield determined using a multivariate calibration model constructed by combining spectroscopic analysis with partial least-squares regression modeling. The ternary extrudates exhibited a dissolution profile similar to that of the 1:2 prepared eutectic, suggesting a physical distribution (or suspension) of the in situ synthesized eutectic contents within the polymeric matrix.

Integrative Salt Selection and Formulation Optimization: Perspectives of Disproportionation and Microenvironmental pH Modulation.

We report a novel utilization of a pH modifier as a disproportionation retardant in a tablet formulation. The drug molecule of interest has significant bioavailability challenges that require solubility enhancement. In addition to limited salt/cocrystal options, disproportionation of the potential salt(s) was identified as a substantial risk. Using a combination of Raman spectroscopy with chemometrics and quantitative X-ray diffraction in specially designed stress testing, we investigated the disproportionation phenomena. The learnings and insight drawn from crystallography drove the selection of the maleate form as the target API. Inspired by the fumarate form's unique stability and solubility characteristics, we used fumaric acid as the microenvironmental pH modulator. Proof-of-concept experiments with high-risk (HCl) and moderate-risk (maleate) scenarios confirmed the synergistic advantage of fumaric acid, which interacts with the freebase released by disproportionation to form a more soluble species. The resultant hemifumarate helps maintain the solubility at an elevated level. This work demonstrates an innovative technique to mediate the solubility drop during the "parachute" phase of drug absorption using compendial excipients, and this approach can potentially serve as an effective risk-mitigating strategy for salt disproportionation.

Vinyl Ether Maleic Acid Polymers: Tunable Polymers for Self-Assembled Lipid Nanodiscs and Environments for Membrane Proteins.

Native lipid bilayer mimetics, including those that use amphiphilic polymers, are important for the effective study of membrane-bound peptides and proteins. Copolymers of vinyl ether monomers and maleic anhydride were developed with controlled molecular weights and hydrophobicity through reversible addition-fragmentation chain-transfer polymerization. After polymerization, the maleic anhydride units can be hydrolyzed, giving dicarboxylates. The vinyl ether and maleic anhydride copolymerized in a close to alternating manner, giving essentially alternating hydrophilic maleic acid units and hydrophobic vinyl ether units along the backbone after hydrolysis. The vinyl ether monomers and maleic acid polymers self-assembled with lipids, giving vinyl ether maleic acid lipid particles (VEMALPs) with tunable sizes controlled by either the vinyl ether hydrophobicity or the polymer molecular weight. These VEMALPs were able to support membrane-bound proteins and peptides, creating a new class of lipid bilayer mimetics.

A randomized crossover trial assessing denture adhesive for prevention of food infiltration among partial denture wearers.

PURPOSE: This two-treatment, four-period, double-blind, randomized controlled crossover trial assessed the ability of two denture adhesives, both applied with a thin nozzle in a continuous application pattern, to prevent food infiltration beneath partial dentures. METHODS: Participants with mandibular partial dentures and a history of food particle infiltration were enrolled. All participants used both an optimized calcium/zinc partial salt of polyvinyl methyl ether/maleic acid (PVM/MA) denture adhesive and a calcium/sodium partial salt of PVM/MA test denture adhesive, twice each, throughout four study periods, according to a randomly assigned sequence. At each visit, participants underwent two assessments: once with no denture adhesive (baseline) and once with denture adhesive, 1 hour after adhesive application. For each assessment, participants ate one-half of the top of a poppy seed muffin, and a dental professional counted the seeds retained on the denture and mucosa, which was the primary variable. The change-from-baseline comparison was made for each treatment separately using a paired t-test or Wilcoxon Signed Rank test depending on the normality of the data. A between-treatment comparison for the change from baseline was performed using a crossover ANCOVA with treatment and period as fixed effects and participant as a random effect. The baseline poppy seed count was used as a covariate. RESULTS: 30 participants were enrolled; 29 completed the trial. Both denture adhesives achieved statistically significantly fewer retained seeds versus baseline (P< 0.001). The calcium/zinc adhesive reduced the seed count from baseline by 85.9% (6.18 vs 0.86), and the calcium/ sodium adhesive reduced seed count by 76.6% (6.04 vs 1.43). Comparing the two denture adhesives, the reduction in seed count from baseline was statistically significantly greater for the calcium/zinc adhesive versus the calcium/sodium formulation (P= 0.008). CLINICAL SIGNIFICANCE: These results support the recommendation of denture adhesive use for the prevention of food infiltration beneath partial dentures, with optimized calcium/zinc denture adhesive showing the greatest prevention benefit.

The Enhancement Origin of Antioxidant Property of Carboxylated Lignin Isolated from Herbaceous Biomass Using the Maleic Acid Hydrotropic Fractionation.

Lignin is endowed with antioxidant activity due to its diverse chemical structure. It is necessary to explore the relationship between antioxidant activity and the chemical structure of the lignin to develop its high-value utilization. Herein, we employed maleic acid (MA) as a hydrotropic agent to preferably isolate the lignin from distinct herbaceous sources (wheat straw and switchgrass) under atmospheric pressure conditions. The resultant acid hydrotropic lignin (AHL) isolated from wheat straw exhibited high radical scavenging rates, up to 98% toward DPPH and 94% toward ABTS. Further investigations indicated that during the MA hydrotropic fractionation (MAHF) process, lignin was carboxylated by MA at γ-OH of the side-chain, providing additional antioxidant activity from the carboxy group. It was also found that the radical scavenging rate of AHL has a positive correlation with carboxyl, phenolic hydroxyl contents, and the S-G (syringyl-guaiacyl) ratio, which could be realized by increasing the MAHF severity. Overall, this work underlies the enhancement origin of the antioxidant property of lignin, which will facilitate its application in biological fields as an efficient, cheap, and renewable antioxidant additive.

The Synergetic Reduction of the Condensation Degree of Dissolved Lignin (DL) during the Refining Process of Wheat Straw Biomass Based on the MA/O3 System.

Lignin, a natural pol2ymer with a complex structure that is difficult to separate, is prone to C-C bond condensation during the separation process. To reduce the condensation of lignin, here, a novel method is proposed for separating the components by using a combination of maleic acid (MA)/ozone (O3) to co-treat wheat straw. The removal of lignin, glucan, and xylan was 38.07 ± 0.2%, 31.44 ± 0.1%, and 71.98 ± 0.1%, respectively, under the conditions of ball-milling of wheat straw for 6 h, reaction temperature of 60 °C, and O3 holding time of 9 min. Lignin-rich solutions were collected to extract the dissolved lignin (DL) after washing the treated samples. The DL obtained under MA/O3 conditions had a carboxyl group (-COOH) content of 2.96 mmol/g. The carboxyl group of MA underwent esterification with the hydroxyl group (-OH) at the γ position of lignin and O3 reacted on the positions of the lignin side chain or the phenolic ring, resulting in a break in the side chain and the opening of the phenolic ring to introduce the carboxyl group. The 2D-HSQC-NMR results revealed that the phenolic ring-opening reaction of lignin in the presence of O3 was essentially free of ß-ß and ß-5 condensation bonds.

Torsemide Crystalline Salts with a Significant Spring-Parachute Effect.

Torsemide is a long acting pyridine sulfonylurea diuretic. Torsemide hydrochloride is widely used now, there are only a few organic acid salts reported. Cocrystallization with organic acids is an effective way to improve its solubility. Here, we reported maleate and phthalate of torsemide, in which the organic acid lost a proton transferring to the pyridine of torsemide, and torsemide interacted with organic acid through N+ - H⋯O- hydrogen bond to form salts crystal. Surprisingly, maleate showed a clear "spring" pattern in apparent solubility, whereas phthalate had a "spring-parachute" effect. Both crystalline salts kept a higher solubility than torsemide without falling. The "spring-parachute" effect of crystalline salts promoted rapid dissolution of torsemide and kept a high concentration, thereby increasing its bioavailability.

Lipid packing is disrupted in copolymeric nanodiscs compared with intact membranes.

Discoidal lipid-protein nanoparticles known as nanodiscs are widely used tools in structural and membrane biology. Amphipathic, synthetic copolymers have recently become an attractive alternative to membrane scaffold proteins for the formation of nanodiscs. Such copolymers can directly intercalate into, and form nanodiscs from, intact membranes without detergents. Although these copolymer nanodiscs can extract native membrane lipids, it remains unclear whether native membrane properties are also retained. To determine the extent to which bilayer lipid packing is retained in nanodiscs, we measured the behavior of packing-sensitive fluorescent dyes in various nanodisc preparations compared with intact lipid bilayers. We analyzed styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and polymethacrylate (PMA) as nanodisc scaffolds at various copolymer-to-lipid ratios and temperatures. Measurements of Laurdan spectral shifts revealed that dimyristoyl-phosphatidylcholine (DMPC) nanodiscs had increased lipid headgroup packing compared with large unilamellar vesicles (LUVs) above the lipid melting temperature for all three copolymers. Similar effects were observed for DMPC nanodiscs stabilized by membrane scaffolding protein MSP1E1. Increased lipid headgroup packing was also observed when comparing nanodiscs with intact membranes composed of binary mixtures of 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and di-palmitoyl-phosphocholine (DPPC), which show fluid-gel-phase coexistence. Similarly, Laurdan reported increased headgroup packing in nanodiscs for biomimetic mixtures containing cholesterol, most notable for relatively disordered membranes. The magnitudes of these ordering effects were not identical for the various copolymers, with SMA being the most and DIBMA being the least perturbing. Finally, nanodiscs derived from mammalian cell membranes showed similarly increased lipid headgroup packing. We conclude that nanodiscs generally do not completely retain the physical properties of intact membranes.

Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration.

Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein-protein and protein-lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins.

Structural Insights into Polymer-Bounded Lipid Nanodiscs.

Membrane proteins are an essential part of signaling and transport processes and are targeted by multiple drugs. To isolate and investigate them in their native state, polymer-bounded nanodiscs have become valuable tools. In this study, we investigate the lipid model system dimyristoyl-phosphocholine (DMPC) with the nanodisc-forming copolymers styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA). Using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we studied the influence of polymer concentration and temperature on the nanodisc structure. In Tris buffer, the size of nanodiscs formed with SMA is smaller compared to DIBMA at the same polymer ratio. In both cases, the size decreases monotonically with increasing polymer concentration, and this effect is more pronounced when using SMA. Measurements at temperatures (T) between 5 and 30 °C in phosphate buffer showed an incomplete solubilization at high T even at polymer/lipid ratios above that required for complete lipid solubilization. For DIBMA, the nanodiscs developed at lower temperatures are stable and the net repulsion increases, while for SMA, the individual nanodiscs possess smaller sizes and are less affected by T. However, using DLS, one can observe SMA agglomerates at low T. Interestingly, for both polymers, no drastic changes of the observable parameters (radius and bilayer thickness) are seen upon cooling, which would indicate a sharp (first-order) phase transition from liquid-crystalline to gel, but only gradual changes. Hence, we conclude that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer. These results can pave the way toward the development of better protocols for studying membrane proteins stabilized in this type of membrane mimics.

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles.

Different amphiphilic co-polymers have been introduced to produce polymer-lipid particles with nanodisc structure composed of an inner lipid bilayer and polymer chains self-assembled as an outer belt. These particles can be used to stabilize membrane proteins in solution and enable their characterization by means of biophysical methods, including small-angle X-ray scattering (SAXS). Some of these co-polymers have also been used to directly extract membrane proteins together with their associated lipids from native membranes. Styrene/maleic acid and diisobutylene/maleic acid are among the most commonly used co-polymers for producing polymer-lipid particles, named SMALPs and DIBMALPs, respectively. Recently, a new co-polymer, named Glyco-DIBMA, was produced by partial amidation of DIBMA with the amino sugar N-methyl-d-glucosamine. Polymer-lipid particles produced with Glyco-DIBMA, named Glyco-DIBMALPs, exhibit improved structural properties and stability compared to those of SMALPs and DIBMALPs while retaining the capability of directly extracting membrane proteins from native membranes. Here, we characterize the structure and lipid composition of Glyco-DIBMALPs produced with either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Glyco-DIBMALPs were also prepared with mixtures of either POPC or DMPC and cholesterol at different mole fractions. We estimated the lipid content in the Glyco-DIBMALPs and determined the particle structure and morphology by SAXS. We show that the Glyco-DIBMALPs are nanodisc-like particles whose size and shape depend on the polymer/lipid ratio. This is relevant for designing nanodisc particles with a tunable diameter according to the size of the membrane protein to be incorporated. We also report that the addition of >20 mol % cholesterol strongly perturbed the formation of Glyco-DIBMALPs. Altogether, we describe a detailed characterization of the Glyco-DIBMALPs, which provides relevant inputs for future application of these particles in the biophysical investigation of membrane proteins.

SMALPs Are Not Simply Nanodiscs: The Polymer-to-Lipid Ratios of Fractionated SMALPs Underline Their Heterogeneous Nature.

Amphipathic styrene-maleic acid (SMA) copolymers directly solubilize biomembranes into SMA-lipid particles, or SMALPs, that are often regarded as nanodiscs and hailed as a native membrane platform. The promising outlook of SMALPs inspires the discovery of many SMA-like copolymers that also solubilize biomembranes into putative nanodiscs, but a fundamental question remains on how much the SMALPs or SMALP analogues truly resemble the bilayer structure of nanodiscs. This unfortunate ambiguity undermines the utility of SMA or SMA-like copolymers in membrane biology because the structure and function of many membrane proteins depend critically on their surrounding matrices. Here, we report the structural heterogeneity of SMALPs revealed through fractionating SMALPs comprised of lipids and well-defined SMAs via size-exclusion chromatography followed by quantitative determination of the polymer-to-lipid (P/L) stoichiometric ratios in individual fractions. Through the lens of P/L stoichiometric ratios, different self-assembled polymer-lipid nanostructures are inferred, such as polymer-remodeled liposomes, polymer-encased nanodiscs, polymer-lipid mixed micelles, and lipid-doped polymer micellar aggregates. We attribute the structural heterogeneity of SMALPs to the microstructure variations amongst individual polymer chains that give rise to their polydisperse detergency. As an example, we demonstrate that SMAs with a similar S/MA ratio but different chain sizes participate preferentially in different polymer-lipid nanostructures. We further demonstrate that proteorhodopsin, a light-driven proton pump solubilized within the same SMALPs is distributed amongst different self-assembled nanostructures to display different photocycle kinetics. Our discovery challenges the native nanodisc notion of SMALPs or SMALP analogues and highlights the necessity to separate and identify the structurally dissimilar polymer-lipid particles in membrane biology studies.

Conformational trapping of an ABC transporter in polymer lipid nanoparticles.

ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC-SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866-SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866-SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866-SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.

Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids.

One of the biggest challenges in membrane protein (MP) research is to secure physiologically relevant structural and functional information after extracting MPs from their native membrane. Amphipathic polymers represent attractive alternatives to detergents for stabilizing MPs in aqueous solutions. The predominant polymers used in MP biochemistry and biophysics are amphipols (APols), one class of which, styrene maleic acid (SMA) copolymers and their derivatives, has proven particularly efficient at MP extraction. In order to examine the relationship between the chemical structure of the polymers and their ability to extract MPs from membranes, we have developed two novel classes of APols bearing either cycloalkane or aryl (aromatic) rings, named CyclAPols and ArylAPols, respectively. The effect on solubilization of such parameters as the density of hydrophobic groups, the number of carbon atoms and their arrangement in the hydrophobic moieties, as well as the charge density of the polymers was evaluated. The membrane-solubilizing efficiency of the SMAs, CyclAPols, and ArylAPols was compared using as models (i) two MPs, BmrA and a GFP-fused version of LacY, overexpressed in the inner membrane of Escherichia coli, and (ii) bacteriorhodopsin, naturally expressed in the purple membrane of Halobacterium salinarum. This analysis shows that, as compared to SMAs, the novel APols feature an improved efficiency at extracting MPs while preserving native protein-lipid interactions.

Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment.

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.

Formation of styrene maleic acid lipid nanoparticles (SMALPs) using SMA thin film on a substrate.

Despite the important role of membrane proteins in biological function and physiology, studying them remains challenging because of limited biomimetic systems for the protein to remain in its native membrane environment. Cryo electron microscopy (Cryo-EM) is emerging as a powerful tool for analyzing the structure of membrane proteins. However, Cryo-EM and other membrane protein analyses are better studied in a native lipid bilayer. Although traditional, mimetic systems have disadvantages that limit their use in the study of membrane proteins. As an alternative, styrene-maleic acid copolymers are used to form nanoparticles with POPC:POPG lipids. Traditional characterization of these styrene maleic acid lipid nanoparticles (SMALPs) includes dynamic light scattering (DLS), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and transmission electron microscopy (TEM). In this study a new method was developed that utilizes SMALPs using a styrene-maleic acid copolymer (SMA) thin film on a TEM grid, acting as a substrate. By directly adding POPC:POPG lipid vesicles to the SMA coated grid SMALPs can be formed, visualized, and characterized by TEM without the need to make them in solution prior to imaging. We envision these functionalized grids could aid in single particle specimen preparation, increasing the efficiency of structural biology and biophysical techniques such as Cryo-EM.