Styrene-maleic Acid Research Articles

Characterizing the Structure of Styrene-Maleic Acid Copolymer-Lipid Nanoparticles (SMALPS) using Raft Polymerization for Membrane Protein Spectroscopic Studies

Membrane proteins are necessary for the function of a variety of biological systems. They assist in transporting ions across membranes, acting as receptors affecting down-stream signaling pathways and providing structure and assembly to cells. The function of a majority of proteins are known, but little is known about their structure. Successful analysis of membrane proteins using biophysical techniques is difficult to accomplish due to protein denaturization. Membrane proteins must be in a native environment once extracted, which poses a challenge to extract the protein to analyze using spectroscopic techniques. Membrane mimetic systems such as detergent micelles, bicelles, nanodiscs, and lipodisqs have been shown to be successful for studying membrane proteins, but each system has its limitations. Polymers have recently become a promising mechanism for stabilizing membrane proteins and form a homogeneous membrane mimetic system. Recent studies have shown that a stabilizing polymer, styrene-maleic acid (StMA), can be synthesized using reversible-addition fragmentation chain transfer polymerization (RAFT) styrene-maleic acid copolymer-lipid nanoparticles (SMALPs). This homogeneous SMALP system was characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM) and Electron Paramagnetic Resonance (EPR) spectroscopy. Three different lipid environments were titrated with styrene-maleic acid at a ratio of 3:1 (St:MA). This promising membrane mimetic system forms homogeneous SMALPs at a weight ratio of 1:1.5 (lipid:StMA) with nanoparticles ∼10 nm. V156C, a residue that lies in the hydrophobic interior of KCNQ1-Voltage Sensor Domain (Q1-VSD), was successfully incorporated into SMALPs. Continuous Wave- EPR line shape analysis shows two components of the spin label; one shows line shape broadening, indicating a lower rigid motional component and the second indicates a faster component. This study will open paths for researchers studying membrane proteins in more native membrane mimetic using biophysical techniques.

Structure of the Alternative Complex III in a Supercomplex with Cytochrome Oxidase

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1–3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4–7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron–sulfur molybdoenzyme (CISM) superfamily8, including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11–14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 A resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe–4S] cluster, a [4Fe–4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer. The structure of alternative complex III, a key enzyme in the bacterial electron transport chain, is reported both alone and as a supercomplex with an aa3-type cytochrome c oxidase.

Biological Membrane Solubilization by Styrene-Maleic Acid Copolymers: Importance of Polymer Length

Biological Membrane Solubilization by Styrene-Maleic Acid Copolymers: Importance of Polymer Length

Detergent-free solubilization of human Kv channels expressed in mammalian cells.

Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

Raloxifene nano-micelles effect on triple-negative breast cancer is mediated through estrogen receptor-β and epidermal growth factor receptor

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that differs in progression, recurrence, and prognosis from other forms of breast cancer. The heterogeneity of TNBC has remained a challenge as no targeted therapy is currently available. Previously, we and others have demonstrated that raloxifene, a selective oestrogen receptor modulator, was also acting independently of the oestrogen receptor-α. However, raloxifene is characterised by a low bioavailability in vivo. Thus, we encapsulated raloxifene into a styrene-maleic acid (SMA) micelle to improve its pharmacokinetics. The micellar raloxifene had higher cytotoxicity when compared to the free formulation, promoted a higher cellular uptake and affected critical signalling pathways. Furthermore, SMA-raloxifene reduced TNBC tumour growth more efficiently than free raloxifene. Finally, we showed that this effect was partially mediated through oestrogen receptor-β. In conclusion, we have provided new insight into the role of raloxifene nanoformulation in improving the management of TNBC.

Morphology and Mechanical Properties of Polyamide 6/Acrylonitrile-Butadiene-Styrene Blends Containing Carbon Nanotubes

The blends of polyamide 6/acrylonitrile-butadiene-styrene (PA6/ABS), with added styrene-maleic acid copolymer (SMA) compatibilizer, were prepared through melt mixing in an internal mixer. The effects of blend composition and various process conditions, as well as the addition of multi-wall carbon nanotubes (MWCNTs) to the blends, on the morphology and mechanical properties were investigated. The morphology of the blends and blend nanocomposites were observed by scanning electron microscopy (SEM) and analyzed using an image analysis technique. The mechanical behavior of the blends was investigated by tensile and also impact testing. The results showed that the blend composition as well as the processing conditions significantly affected the morphology and mechanical properties of the PA6/ABS blends. Among the various compositions, the blend with 36 wt.% of ABS and 4 wt.% of SMA compatibilizer exhibited the best mechanical properties. Comparing various speeds and times of mixing, it was found that less mixing speed and longer mixing times resulted in the favorable morphology and conditions for achievement of the desired toughness for the polyamide 6. By adding different amounts of MWCNTs to the blends, it was found that the presence of the carbon nanotubes changed the viscosity of the resulting nanocomposite and thus changed the morphology. These nanocomposites also showed an improvement in mechanical properties. The MWCNTs acted as a second compatibilizer, resulting in a synergistic effect on the mechanical properties of the PA6/ABS blend nanocomposites.

Molecular snapshots of dynamic membrane-bound metabolons.

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

Styrene maleic acid copolymer induces pores in biomembranes.

We investigated the interactions between styrene-maleic acid (SMA) copolymers and phospholipid bilayers, using confocal microscopy and surface acoustic wave resonance (SAR) sensing. For the first time we experimentally observed and followed pore formation by SMA copolymers in intact supported bilayers and unilamellar vesicles, showing that fluorescein, a water-soluble organic compound with a mean diameter of 6.9 Å, can traverse the membrane. Our findings are in agreement with recent theoretical predictions, which suggested that SMA copolymers may insert along the plane of the bilayer to form stable toroidal pores.

Correction: Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers.

[This corrects the article DOI: 10.1371/journal.pone.0206692.].

Examining the stability of membrane proteins within SMALPs

Amphipathic co-polymers such as styrene-maleic acid (SMA) have gained popularity over the last few years due to their ability and ease of use in solubilising and purifying membrane proteins in comparison to conventional methods of extraction such as detergents. SMA2000 is widely used for membrane protein studies and is considered as the optimal polymer for this technique. In this study a side-by-side comparison of SMA2000 with the polymer SZ30010 was carried out as both these polymers have similar styrene:maleic acid ratios and average molecular weights. Ability to solubilise, purify and stabilise membrane proteins was tested using three structurally different membrane proteins. Our results show that both polymers can be used to extract membrane proteins at a comparable efficiency to conventional detergent dodecylmaltoside (DDM). SZ30010 was found to give a similar protein yield and, SMALP disc size as SMA2000, and both polymers offered an increased purity and increased thermostability compared to DDM. Further investigation was conducted to investigate SMALP sensitivity to divalent cations. It was found that the sensitivity is polymer specific and not dependent on the protein encapsulated. Neither is it affected by the concentration of SMALPs. Larger divalent cations such as Co2+ and Zn2+ resulted in an increased sensitivity.

Characterizing the structure of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for membrane protein spectroscopic studies.

Membrane proteins play an important role in maintaining the structure and physiology of an organism. Despite their significance, spectroscopic studies involving membrane proteins remain challenging due to the difficulties in mimicking their native lipid bilayer environment. Membrane mimetic systems such as detergent micelles, liposomes, bicelles, nanodiscs, lipodisqs have improved the solubility and folding properties of the membrane proteins for structural studies, however, each mimetic system suffers from its own limitations. In this study, using three different lipid environments, vesicles were titrated with styrene-maleic acid (StMA) copolymer leading to a homogeneous SMALP system (∼10 nm) at a weight ratio of 1:1.5 (vesicle: StMA solution). A combination of Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) was used to characterize these SMALPs. We used a controlled synthesis mechanism to synthesize StMA based block copolymers called reversible addition-fragmentation chain transfer polymerization (RAFT) SMALPs. Incorporation of the Voltage Sensor Domain of KCNQ1 (Q1-VSD) into RAFT SMALPs indicates that this is a promising application of this system to study membrane proteins using different biophysical techniques. V165C in Q1-VSD corresponding to the hydrophobic region was incorporated into the SMALP system. Continuous Wave-Electron Paramagnetic Resonance (CW-EPR) line shape analysis showed line shape broadening, exposing a lower rigid component and a faster component of the spin label.

A simple and convenient method for the hydrolysis of styrene-maleic anhydride copolymers to styrene-maleic acid copolymers

Styrene-maleic acid (SMA) copolymers are increasingly gaining attention in the membrane protein field due to their ability to solubilize lipid membranes into discoidal nanoparticles. The copolymers are synthesized as styrene-maleic anhydride (SMAnh), and need to be converted to the free acid form (SMA) before they are capable of solubilizing membranes. This hydrolysis reaction is traditionally performed under rather cumbersome reflux conditions. Here we report an alternative method for the hydrolysis reaction using simple and readily available equipment found in virtually all biochemical laboratories, namely an autoclave. Based on the results we propose an optimum set of standard conditions for the hydrolysis reaction, that should make the method easily accessible to a wide scope of researchers.

Membrane protein nanoparticles: the shape of things to come.

The use of styrene–maleic acid (SMA) for the purification of a wide range of membrane proteins (MPs) from both prokaryotic and eukaryotic sources has begun to make an impact in the field of MP biology. This method is growing in popularity as a means to purify and thoroughly investigate the structure and function of MPs and biological membranes. The amphiphilic SMA copolymer can effectively extract MPs directly from a native lipid bilayer to form discs ∼10 nm in diameter. The resulting lipid particles, or styrene–maleic acid lipid particles (SMALPs), contain SMA, protein and membrane lipid. MPs purified in SMALPs are able to retain their native structure and, in many cases, functional activity, and growing evidence suggests that MPs purified using SMA have enhanced thermal stability compared with detergent-purified proteins. The SMALP method is versatile and is compatible with a wide range of cell types across taxonomic domains. It can readily be adapted to replace detergent in many protein purification methods, often with only minor changes made to the existing protocol. Moreover, biophysical analysis and structural determination may now be a possibility for many large, unstable MPs. Here, we review recent advances in the area of SMALP purification and how it is affecting the field of MP biology, critically assess recent progress made with this method, address some of the associated technical challenges which may remain unresolved and discuss opportunities for exploiting SMALPs to expand our understanding of structural and functional properties of MPs.

Analysis of styrene maleic acid alternating copolymer supramolecular assemblies in solution by small angle X-ray scattering

Over the past decade, Styrene Maleic Acid alternating copolymers (SMA) have gained interest as an alternative to detergent solubilization for the isolation of integral membrane proteins. The formation of SMA lipid particles (SMALPs) presents a novel opportunity to isolate the proximal membrane environment, encompassing and throughout membrane proteins in vitro. Neither the organization or structure of SMAs in an aqueous buffer nor the mechanism by which SMA transforms the membrane bilayer into a SMALP is known. This study investigates the shape and size of diverse SMA polymer complexes/aggregates in solution at various pH, ionic strength, SMA concentration and temperature, analyzed by small angle X-ray scattering. It is clear that SMAs of differing physicochemical properties (styrene to maleic acid ratio, length of copolymer fragments and functionalization) display highly variable sizes/shapes in solution over a range of environmental conditions. The SMA supramolecular aggregates exhibit similar prolate ellipsoidal geometry of varying size, dependent on the degree of hydrophobicity of the SMA copolymer. At elevated temperature, particles composed of SMAs enriched in styrene increase in both radius of gyration and maximum particle diameter. Interestingly, we observe a correlation between the SMALP dimensions and that of native membranes. Future work will investigate if there may be a complimentary relationship between SMA aggregate dimensions and bilayer thickness (and/or protein transmembrane domain thickness), similar to what has been observed for tandem facial amphiphiles.

The lipid environment of Escherichia coli Aquaporin Z

In this study, we have investigated the lipids surrounding AqpZ, and the effects of a destabilizing mutation W14A (Schmidt and Sturgis, 2017) on lipid protein interactions. In a first approach, we used Styrene Maleic Acid copolymer to prepare AqpZ containing nanodiscs, and these were analyzed for their lipid content, investigating both the lipid head-group and acyl-chain compositions. These results were complemented by native mass spectrometry of purified AqpZ in the presence of lipids, to give insights of variations in lipid binding at the surface of AqpZ. In an effort to gain molecular insights, to aid interpretation of these results, we performed a series of coarse grained molecular dynamics simulations of AqpZ, in mixed lipid membranes, and correlated our observations with the experimental measurements. These various results are then integrated to give a clearer picture of the lipid environment of AqpZ, both in the native membrane, and in lipid nanodiscs. We conclude that AqpZ contains a lipid binding-site, at the interface between the monomers of the tetramer, that is specific for cardiolipin. Almost all the cardiolipin, in AqpZ containing nanodiscs, is probably associated with this site. The SMA 3:1 nanodiscs we obtained contain a rather high proportion of lipid, and in the case of nanodiscs containing AqpZ cardiolipin is depleted. This is possibly because, in the membrane, there is little cardiolipin not associated with binding sites on the surface of the different membrane proteins. Surprisingly, we see no evidence for lipid sorting based on acyl chain length, even in the presence of a large hydrophobic mismatch, suggesting that conformational restrictions are energetically less costly than lipid sorting.

Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers.

As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently developed detergent-free styrene-maleic acid (SMA) co-polymer system offers an alternative approach that may overcome these disadvantages. In this study, we used the detergent free system to purify MraY from Bacillus subtilis. This allowed efficient extraction of MraY that was heterologously produced in Escherichia coli membranes into SMA-wrapped nanodiscs. The purified MraY embedded in these nanodiscs (SMA-MraY) was comparable to the micellar MraY extracted with a conventional detergent (DDM) with regard to the yield and the purity of the recombinant protein but required significantly less time. The predominantly alpha-helical secondary structure of the protein in SMA-wrapped nanodiscs was also more stable against heat denaturation compared to the micellar protein. Thus, this detergent-free system is amenable to extract MraY efficiently and effectively while maintaining the biophysical properties of the protein. However, the apparent activity of the SMA-MraY was reduced compared to that of the detergent-solubilized protein. The present data indicates that this is caused by a lower accessibility of the enzyme in SMA-wrapped nanodiscs towards its polyisoprenoid substrate.

Evaluation of commercially available styrene-co-maleic acid polymers for the extraction of membrane proteins from spinach chloroplast thylakoids

Solubilization of membrane proteins by poly(styrene-co-maleic acid) salts (pSMA-S) has significant potential for membrane protein studies. This approach provides an opportunity to overcome many disadvantages associated with a traditional detergent-based technique including protein denaturation and displacement of boundary lipids which may offer both structural and functional stability to membrane proteins. Thylakoid membranes (TMs) from photosynthetic organisms are well studied protein-rich membranes that host several multi-subunit protein complexes associated with oxygenic photosynthesis. These protein complexes are important for applied photosynthesis and by being extracted and purified they can be used in the near future for direct energy conversion. In this study, we used spinach TMs isolated from purified intact chloroplasts to systematically test the solubilization efficacy of 12 commercially available styrene-maleic acid (SMA) copolymers that vary in size, styrene-to-maleic acid molar ratio, and type of ester group. The efficacy of these pSMA-S to solubilize protein-containing biomembranes was evaluated via quantification of protein and chlorophyll content in the resulting SMA Lipid Particles (SMALPs). In addition, the extracted polymer-lipid-protein complexes were studied by low temperature fluorescence, sodium dodecyl sulfate and clear native polyacrylamide gel electrophoresis (SDS- and CN-PAGE), and immunoblot analysis. Our results indicate considerable variability in the solubilization efficacy of commercially available pSMA-S with at least 5 polymer formulations being able to efficiently extract membrane proteins from TMs. These 5 SMA copolymers may also be effective in extraction of membrane proteins from other biomembranes.

Generation of membrane proteins in polymer-based lipoparticles as flow cytometry antigens

A major obstacle in the study of membrane proteins is the challenge in generating purified targets in a stable and native conformation. Potentially destabilizing detergents are often used to extract targets out of their native membranes. One approach to overcome these destabilization issues is to present targets in a lipid bilayer environment. While numerous lipid bilayer presentation approaches such as Nanodiscs and liposomes have been used through the years, most still require a detergent purified protein intermediate. Styrene-maleic acid lipoparticles (SMALPs) have been developed to directly solubilize membranes without the use of detergents and present membrane proteins in a native lipid bilayer environment. These discoidal lipid bilayer particles have been evaluated on several membrane protein classes (e.g. ion channels, transporters) and applications (e.g. spectroscopy, electron microscopy). Here, cannabinoid receptor 1 (CB1), a class A G-protein coupled receptor, was incorporated into a SMALP (CB1-SMALP) and shown to retain its native fold through binding studies with a CB1-specific conformational antibody. The binding studies were conducted in yeast and mammalian display formats using fluorescence-activated cell sorting (FACS). Compared to detergent preparations, CB1-SMALP also exhibited a marked increase in thermostability. This study provides the framework for a platform to generate full-length membrane protein screening antigens for flow cytometry-based studies.

Collisional lipid exchange among DIBMA-encapsulated nanodiscs (DIBMALPs)

Copolymers of diisobutylene/maleic acid (DIBMA), styrene/maleic acid (SMA), and styrene/maleimide (SMI) solubilise membrane proteins and surrounding lipids directly from artificial and biological membranes to form polymer-bounded nanodiscs. Although they preserve a lipid-bilayer core, these nanodiscs are much more dynamic than other membrane mimics, as we have recently demonstrated for two SMA variants. Here, we used time-resolved Förster resonance energy transfer spectroscopy to quantify the kinetics and unravel the mechanisms of lipid transfer among DIBMA-encapsulated nanodiscs (DIBMALPs), with particular emphasis on the role of Coulombic repulsion, which is modulated by ionic strength and nanodisc size. Our results show that DIBMALPs exchange lipids through both binary and ternary collisions on the timescale of seconds to minutes under typical experimental conditions. By contrast, nanodiscs bounded by SMA polymers exchange lipids considerably faster through binary collisions, whereas vesicles and nanodiscs based on membrane scaffold proteins exchange lipids much more slowly through lipid monomer diffusion. As DIBMALPs are polyanionic, collisional lipid transfer is substantially accelerated by screening of Coulombic repulsion caused by increasing ionic strength or nanodisc size. Analysis of the ionic-strength dependence in terms of an extended version of the Debye–Hückel equation suggests an effective DIBMALP charge number of z = −47, which is considerably larger than that previously determined for nanodiscs bounded by more hydrophobic SMA polymers.

Synthesis of imprinted styrene-maleic acid functionalized resin for enantio-selective extraction of R-amphetamine

This article presents the development of an enantio-selective polymeric resin based on cross-linked styrene-maleic acid copolymer for chiral recognition of R-amphetamine and efficient optical resolution of (±)-amphetamine racemate. In the beginning, a polymerizable R-amphetamine-maleimide derivative was synthesized and investigated using elemental analysis, FTIR, and NMR spectroscopy to affirm the chemical structure. The chiral maleimide derivative was then copolymerized with styrene and divinylbenzene via free radical polymerization and the obtained polymeric resin was agitated with sodium hydroxide followed by HCl to expel the R-amphetamine template molecules out of the polymer texture. The obtained molecularly imprinted polymer was characterized using SEM, FTIR and EDX spectra then evaluated for selective removal of R-amphetamine under various conditions. The results indicated that the maximum uptake was achieved at pH 7 and the adsorption was performed in accordance with the Langmuir model with a maximum capacity of 210 ± 1 mg/g. In addition, the optical separation was carried out using a column and the results indicated approximate enantiomeric excess values of 76.5% and 56.5% within both supernatant and eluant solution referring to S- and R-amphetamine approximately, respectively.