Conformations of membrane human immunodeficiency virus (HIV-1) envelope glycoproteins solubilized in Amphipol A18 lipid-nanodiscs.

Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.

Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel.

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

Biochemical interactions between the Atm1-like transporter from Novosphingobium aromaticivorans and heavy metals.

Novosphingobium aromaticivorans has the ability to survive in harsh environments by virtue of its suite of iron-containing oxygenases that biodegrade an astonishing array of aromatic compounds. It is also resistant to heavy metals through Atm1, an ATP-binding cassette protein that mediates active efflux of heavy metals conjugated to glutathione. However, Atm1 orthologues in higher organisms have been implicated in the intracellular transport of organic iron complexes. Our hypothesis suggests that the ability of Atm1 to remove heavy metals is related to the need for regulated iron handling in N. aromaticivorans to support high oxygenase activity. Here we provide the first data demonstrating a direct interaction between an iron-porphyrin compound (hemin) and NaAtm1. Hemin displayed considerably higher binding affinity and lower EC50 to stimulate ATP hydrolysis by Atm1 than Ag-GSH, GSSG or GSH, established substrates of the transporter. Co-incubation of NaAtm1 and hemin with Ag-GSH in ATPase assays revealed a non-competitive interaction, indicating distinct binding sites on NaAtm1 and this property was reinforced using molecular docking analysis. Our data suggests that NaAtm1 has considerable versatility in transporting organic conjugates of metals and that this versatility enables it to play roles in detoxification processes for toxic metals and in homeostasis of iron. The ability to play these distinct roles is enabled by the plasticity of the substrate binding site within the central cavity of NaAtm1.

A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles.

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

Native nanodiscs formed by styrene maleic acid copolymer derivatives help recover infectious prion multimers bound to brain-derived lipids.

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.

Polymer Nanodiscs and Their Bioanalytical Potential.

Membrane proteins (MPs) play a pivotal role in cellular function and are therefore predominant pharmaceutical targets. Although detailed understanding of MP structure and mechanistic activity is invaluable for rational drug design, challenges are associated with the purification and study of MPs. This review delves into the historical developments that became the prelude to currently available membrane mimetic technologies before shining a spotlight on polymer nanodiscs. These are soluble nanosized particles capable of encompassing MPs embedded in a phospholipid ring. The expanding range of reported amphipathic polymer nanodisc materials is presented and discussed in terms of their tolerance to different solution conditions and their nanodisc properties. Finally, the analytical scope of polymer nanodiscs is considered in both the demonstration of basic nanodisc parameters as well as in the elucidation of structures, lipid-protein interactions, and the functional mechanisms of reconstituted membrane proteins. The final emphasis is given to the unique benefits and applications demonstrated for native nanodiscs accessed through a detergent free process.

Structure and activity of lipid bilayer within a membrane-protein transporter.

Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å resolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of the AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner leaflet chains are packed in a hexagonal array, whereas the outer leaflet has highly irregular but ordered packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. We suggest that the lipid bilayer supports and harmonizes peristaltic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost in crystal structures after detergent-solubilization. Our detergent-free system preserves lipid-protein interactions for visualization and should be broadly applicable.

Styrene-maleic acid copolymer-encapsulated carbon monoxide releasing molecule-2 (SMA/CORM-2) suppresses proliferation, migration and invasion of colorectal cancer cells in vitro and in vivo.

Although increasing evidence have confirmed that carbon monoxide release molecule-2(CORM-2) plays an active role in the treatment of inflammation and tumors, poor aqueous solubility and short CO-release duration restrict its extensive application. Our previous work synthesized styrene-maleic acid copolymer-encapsulated CORM-2 (SMA/CORM-2) to overcome above-mentioned deficiencies and demonstrated satisfactory effects in colitis. This study is to investigate the function of SMA/CORM-2 on colorectal cancer proliferation and metastasis. CCK-8 experiment is used to clarify the half maximal inhibitory concentration (IC50) of SMA/CORM-2 and to detect cell proliferation. Transwell assay coated with or without matrigel was to detect cell invasion and migration. Western blot was used to detect ß-catenin, AKT, p-AKT, VEGF, MMP-2 and MMP-9 proteins. At last, nude mice xenograft was used to further investigate the anti-tumor effect of SMA/CORM-2 in vivo. After SW480 and C26 cells were treated with 0.5 mg/ml SMA/CORM-2, CRC cells proliferation, migration and invasion were inhibited. In vivo, SMA/CORM-2 treatment remarkably suppressed tumor growth and lung metastasis in nude mice. Furthermore, the expression of ß-catenin, p-AKT, VEGF, MMP-2 and MMP-9 proteins could be down-regulated after SMA/CORM-2 treatment. SMA/CORM-2 exerted both in vitro and in vivo anti-proliferation and anti-metastatic effects, which may yield a novel therapeutic strategy for CRC.

Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer.

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers.

Discovering how membrane proteins recognize signals and passage molecules remains challenging. Life depends on compartmentalizing these processes into dynamic lipid bilayers that are technically difficult to work with. Several polymers have proven adept at separating the responsible machines intact for detailed analysis of their structures and interactions. Styrene maleic acid (SMA) co-polymers efficiently solubilize membranes into native nanodiscs and, unlike amphipols and membrane scaffold proteins, require no potentially destabilizing detergents. Here we review progress with the SMA lipid particle (SMALP) system and its impacts including three dimensional structures and biochemical functions of peripheral and transmembrane proteins. Polymers systems are emerging to tackle the remaining challenges for wider use and future applications including in membrane proteomics, structural biology of transient or unstable states, and discovery of ligand and drug-like molecules specific for native lipid-bound states.

Antitumor activity of raloxifene-targeted poly(styrene maleic acid)-poly (amide-ether-ester-imide) co-polymeric nanomicelles loaded with docetaxel in breast cancer-bearing mice.

Purpose Raloxifene (RA) receptors have over-expressed GPER-positive breast cancer tumors. The purpose of this work was to evaluate the antitumor activity and pharmacokinetic behavior of docetaxel (DTX), loaded in RA-targeted nanomicelles, which were designed to overcome a lack of specific distribution and inadequate DTX concentration in tumor tissues, as well as its cytotoxicity and damage to normal tissues. Methods DTX-loaded RA-targeted poly(styrene maleic acid) (SMA)- poly(amide-ether-esterimide)-poly(ethylene glycol) (PAEEI-PEG) nanomicelles were prepared; then, their antitumor activity and survival rate were studied in MC4-L2 tumors induced in BALB/c mice. The pharmacokinetics of DTX-loaded SMA-PAEEI-PEG-RA micelles was also investigated in comparison with free DTX. Results DTX-loaded SMA-PAEEI-PEG-RA micelles inhibited tumor growth considerably and increased animal survival as compared to free DTX and non-targeted micelles. SMA-PAEEIPEG-RA micelles enhanced significantly the area under the curve (AUC0-∞) 1.3 times as compared to free DTX and reduced clearance (CL) from 410.43 ml/kg.h (for free DTX) to 308.8 ml/kg.h (for SMA-PAEEI-PEG-RA micelles). Volume of distribution (Vdss) was also reduced 1.4 times as compared to free DTX. RA-targeted micelles increased tumor inhibition rate (TIR) 1.3 times and median survival time (MST) >1.5 times compared to free DTX. Percentage increase in life span (%ILS) was also enhanced significantly from 41.66% to >83.33% in MC4-L2 tumor-bearing BALB/c mice. Discussion All studies in this work showed the potential of DTX-loaded SMA-PAEEI-PEG-RA micelles in the treatment of GPER-positive receptor breast cancer tumors.

Indium-111 labeling of high-density lipoprotein-mimicking phospholipid-styrene maleic acid copolymer complexes and its biodistribution in mice.

Discoidal lipid nanoparticles mimicking native high-density lipoproteins (HDL) are promising delivery vehicles of drugs and/or imaging agents. However, little is known about the in vivo biodistribution of such discoidal lipid nanoparticles compared to liposomes, clinically available spherical lipid nanoparticles. Recently, it has been reported that synthetic polymers instead of apolipoproteins can be complexed with phospholipid to form discoidal nanoparticles. In the present study, with the aim of developing phospholipid-synthetic polymer complexes for future clinical applications, the biodistribution of such particles in normal mice was investigated. Lipid nanoparticles comprising 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and styrene maleic acid copolymer (SMA), having sizes similar to native HDL, were prepared using the freeze-sonication method. POPC-SMA complexes remained stable at 37°C for at least 3 days in buffer. By devising ways to avoid detrimental effects accompanied by pH reduction and nonspecific binding of 111 In to SMA, POPC-SMA complexes were successfully labeled with 111 In without affecting particle integrity. The biodistribution of POPC-SMA complexes in normal mice was similar to that of discoidal lipid nanoparticles composed of POPC and apolipoprotein A-I, the major protein constituent of native HDL. Unlike liposomes, the accumulation of POPC-SMA complexes in the spleen was low, suggesting that these complexes are not recognized as foreign substances. To the best of our knowledge, this is the first in vivo study of HDL-mimicking phospholipid-synthetic polymer complexes.

Tuning the size of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for biophysical studies.

Characterization of membrane proteins is challenging due to the difficulty in mimicking the native lipid bilayer with properly folded and functional membrane proteins. Recently, styrene-maleic acid (StMA) copolymers have been shown to facilitate the formation of disc-like lipid bilayer mimetics that maintain the structural and dynamic integrity of membrane proteins. Here we report the controlled synthesis and characterization of StMA containing block copolymers. StMA polymers with different compositions and molecular weights were synthesized and characterized by size exclusion chromatography (SEC). These polymers act as macromolecular surfactants for 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol (POPG) lipids, forming disc like structures of the lipids with the polymer wrapping around the hydrophobic lipid edge. A combination of dynamic light scattering (DLS), solid-state nuclear magnetic resonance (SSNMR) spectroscopy, and transmission electron microscopy (TEM) was used to characterize the size of the nanoparticles created using these StMA polymers. At a weight ratio of 1.25:1 StMA to lipid, the nanoparticle size created is 28+1nm for a 2:1 ratio, 10+1nm for a 3:1 StMA ratio and 32+1nm for a 4:1 StMA ratio independent of the molecular weight of the polymer. Due to the polymer acting as a surfactant that forms disc like nanoparticles, we term these StMA based block copolymers "RAFT SMALPs". RAFT SMALPs show promise as a new membrane mimetic with different nanoscale sizes, which can be used for a wide variety of biophysical studies of membrane proteins.

Encapsulated membrane proteins: A simplified system for molecular simulation.

Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane.

Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer.

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.

Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs.

A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.

Characterization of the annular lipid shell of the Sec translocon.

The bacterial Sec translocase in its minimal form consists of a membrane-embedded protein-conducting pore SecYEG that interacts with the motor protein SecA to mediate the translocation of secretory proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids.

Membrane proteins: is the future disc shaped?

The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology.

Overcoming bottlenecks in the membrane protein structural biology pipeline.

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.