Styrene Maleic Acid Lipid Particles Research Articles

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets

Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and lipid species. Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of these different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. As a first step towards the incorporation of membrane proteins, this work focusses on the conversion of pure lipid SMALPs in GUVs. To evaluate the method, we produced a new form of SMA linked to fluorescein, referred to as FSMA. We demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially as a result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 μM, 4.57 μM, and 3.72 μM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

GPCRs in the round: SMA-like copolymers and SMALPs as a platform for investigating GPCRs

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30–40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and β-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid ‘landscape’ is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of ‘second generation’ SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. 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 tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes.

As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already competent to collect proteins found on mature autophagosomes, including LC3-II. Further, we use styrene-maleic acid lipid particles to reveal the nanoscale organization of protein on LC3-II membranes; ATG9 and LC3-II are each fully integrated into expanding autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.

Immunochemical characterisation of styrene maleic acid lipid particles prepared from Mycobacterium tuberculosis plasma membrane.

Membrane proteins of Mycobacterium tuberculosis (Mtb) can be targeted for the development of therapeutic and prophylactic interventions against tuberculosis. We have utilized the unique membrane-solubilising properties of the styrene maleic acid copolymer <styrene:maleic acid::2:1> (SMA) to prepare and characterise 'styrene maleic acid lipid particles' from the native membrane of Mtb (MtM-SMALPs). When resolved by SDS-PAGE and visualised with coomassie blue, the molecular weights of Mtb membrane (MtM) proteins solubilised by SMA were mostly in the range of 40-70 kDa. When visualised by transmission electron microscopy, MtM-SMALPs appeared as nanoparticles of discrete shapes and sizes. The discoid nanoparticles exhibited a range of diameters of ~10-90 nm, with largest portion (~61%) ranging from 20-40 nm. MtM proteins of a molecular weight-range overlapping with that of MtM-SMALPs were also amenable to chemical cross-linking, revealing protein complex formation. Characterisation using monoclonal antibodies against seven MtM-associated antigens confirmed the incorporation of the inner membrane protein PRA, membrane-associated proteins PstS1, LpqH and Ag85, and the lipoglycan LAM into MtM-SMALPs. Conversely, the peripheral membrane proteins Acr and PspA were nearly completely excluded. Furthermore, although MtM showed an abundance of Con A-binding glycoproteins, MtM-SMALPs appeared devoid of these species. Immune responses of healthcare workers harbouring 'latent TB infection' provided additional insights. While MtM-SMALPs and MtM induced comparable levels of the cytokine IFN-γ, only MtM-SMALPs could induce the production of TNF-α. Antibodies present in the donor sera showed significantly higher binding to MtM than to MtM-SMALPs. These results have implications for the development of MtM-based immunoprophylaxis against tuberculosis.

Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction.

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein-protein and protein-lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor-ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

The effect of polymer end-group on the formation of styrene - maleic acid lipid particles (SMALPs).

A series of block copolymers comprising styrene and maleic acid (SMA) has been prepared using RAFT polymerisation. RAFT often results in a large hydrophobic alkylthiocarbonylthio end group and this work examines its effect on the solution behaviour of the copolymers. SMA variants with, and without, this end group were synthesised and their behaviour compared with a commercially-available random copolymer of similar molecular weight. Dynamic light scattering and surface tension measurements found the RAFT-copolymers preferentially self-assembled into higher-order aggregates in aqueous solution. Small angle neutron scattering using deuterated styrene varients add support to the accepted model that these agreggates comprise a solvent-protected styrenic core with an acid-rich shell. Replacing the hydrophobic RAFT end group with a more hydrophilic nitrile caused differences in the resulting surface activity, attributed to the ability of the adjoining styrene homoblock to drive aggregation. Each of the copolymers formed SMALP nanodiscs with DMPC lipids, which were found to encapsulate a model membrane protein, gramicidin. However, end group variation affected solubilisition of DPPC, a lipid with a higher phase transition temperature. When using RAFT-copolymers terminated with a hydrophobic group, swelling of the bilayer and greater penetration of the homoblock into the nanodisc core occurred with increasing homoblock length. Conversely, commercial and nitrile-terminated RAFT-copolymers produced nanodisc sizes that stayed constant, instead indicating interaction at the edge of the lipid patch. The results highlight how even minor changes to the copolymer can modify the amphiphilic balance between regions, knowledge useful towards optimising copolymer structure to enhance and control nanodisc formation.

How Choice of Model Membrane Affects Protein-Glycosphingolipid Interactions: Insights from Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.

Lipid membrane mimetics and oligomerization tune functional properties of proteorhodopsin

The functional properties of proteorhodopsin (PR) have been found to be strongly modulated by oligomeric distributions and lipid membrane mimetics. This study aims to distinguish and explain their effects by investigating how oligomer formation impacts PR’s function of proton transport in lipid-based membrane mimetic environments. We find that PR forms stable hexamers and pentamers in both E. coli membranes and synthetic liposomes. Compared with the monomers, the photocycle kinetics of PR oligomers is ∼2 and ∼4.5 times slower for transitions between the K and M and the M and N photointermediates, respectively, indicating that oligomerization significantly slows PR’s rate of proton transport in liposomes. In contrast, the apparent pKa of the key proton acceptor residue D97 (pKaD97) of liposome-embedded PR persists at 6.2–6.6, regardless of cross-protomer modulation of D97, suggesting that the liposome environment helps maintain PR’s functional activity at neutral pH. By comparison, when extracted directly from E. coli membranes into styrene-maleic acid lipid particles, the pKaD97 of monomer-enriched E50Q PR drastically increases to 8.9, implying that there is a very low active PR population at neutral pH to engage in PR’s photocycle. These findings demonstrate that oligomerization impacts PR’s photocycle kinetics, while lipid-based membrane mimetics strongly affect PR’s active population via different mechanisms.

The function of BK channels extracted and purified within SMALPs.

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA–PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

Vinca alkaloid binding to P-glycoprotein occurs in a processive manner

A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp; the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit.

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

Advances in the application of fluorescence correlation spectroscopy to study detergent purified and encapsulated membrane proteins.

Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery.

Functional Extraction and Spectroscopic Investigation of Structural Dynamics in E. coli ATP Synthase

ATP synthase is a membrane protein complex that synthesizes adenosine triphosphate (ATP), an integral molecule that provides energy to all forms of life. The complex is comprised of two rotary motors, the soluble F1 sector and the membrane embedded Fo sector. ATP synthesis is catalyzed by conformational changes within F1, which are driven by the rotation of the c‐ring (rotor) of Fo. The Fo motor is powered by the electrochemical gradient of H+ across the cell membrane, and movement of H+ through Fo is facilitated by two aqueous half‐channels within subunit a (stator) and at the rotor‐stator interface. The mechanism of how H+‐driven rotation in Fo is generated is not fully understood. Cryo‐electron microscopy has revealed structures of E. coli F1Fo ATP synthase, but inconsistencies between this structural data and previous cross‐linking data within subunit a suggest the existence of multiple conformations. In order to gain insight into suspected conformational dynamics of the stator, we have utilized site‐directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to observe backbone mobility at several points in subunit a. We demonstrate the feasibility of membrane extraction using mixed micelles of dodecylmaltoside/phosphatidylcholine or styrene maleic acid lipid particles (SMALPs). Furthermore, we found high backbone mobility at sites on the cytoplasmic surface of the subunit a stator.

Biochemical Investigation of Membrane-Bound Cytochrome b5 and the Catalytic Domain of Cytochrome b5 Reductase from Arabidopsis thaliana.

The endoplasmic reticulum (ER) membrane of plant cells contains several enzymes responsible for the biosynthesis of a diverse range of molecules essential for plant growth and holds potential for industrial applications. Many of these enzymes are dependent on electron transfer proteins to sustain their catalytic cycles. In plants, two crucial ER-bound electron transfer proteins are cytochrome b5 and cytochrome b5 reductase, which catalyze the stepwise transfer of electrons from NADH to redox enzymes such as fatty acid desaturases, cytochrome P450s, and plant aldehyde decarbonylase. Despite the high significance of plant cytochrome b5 and cytochrome b5 reductase, they have eluded detailed characterization to date. Here, we overexpressed the full-length membrane-bound cytochrome b5 isoform B from the model plant Arabidopsis thaliana in Escherichia coli, purified the protein employing detergents as well as styrene-maleic acid (SMA) copolymers, and biochemically characterized the protein. The SMA-encapsulated cytochrome b5 exhibits a discoidal shape and the characteristic features of the active heme-bound state. We also overexpressed and purified the soluble domain of cytochrome b5 reductase from A. thaliana, establishing its activity, stability, and kinetic parameters. Further, we demonstrated that the plant cytochrome b5, purified in detergents and styrene maleic acid lipid particles (SMALPs), readily accepts electrons from the cognate plant cytochrome b5 reductase and distant electron mediators such as plant NADPH-cytochrome P450 oxidoreductase and cyanobacterial NADPH-ferredoxin reductase. We also measured the kinetic parameters of cytochrome b5 reductase for cytochrome b5. Our studies are the first to report the purification and detailed biochemical characterization of the plant cytochrome b5 and cytochrome b5 reductase from the bacterial overexpression system.

Magnetically aligned peptoid-based macrodiscs for structure determination of membrane proteins by solid-state NMR

We report on highly alignable, novel lipid mimetic for structural and dynamical studies of membrane proteins by oriented-sample (OS) solid-state NMR which can be used to determine structures of membrane proteins under their native-like conditions without the use of crystallization and cryogenic temperatures. As an alternative to the recently reported discoidal mimetics based on lipoproteins and styrene-maleic acid lipid particles, we have developed magnetically orientable discs of ca. 20-30 nm in diameter based on 15-mer peptoid constructs.

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.

Detergent-Free Membrane Protein Purification Using SMA Polymer.

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those.