Styrene-maleic Acid Research Articles

Structure retention of silica gel-encapsulated bacteriorhodopsin in purple membrane and in lipid nanodiscs

The integral membrane protein, bacteriorhodopsin (BR) was encapsulated in sol-gel derived porous silica gel monoliths in native purple membrane (BR-PM) and synthetic lipid nanodisc (BR nanodisc) environments. BR nanodiscs were synthesized by solubilizing purple membrane in discoidal phospholipid bilayer stabilized by amphipathic Styrene-Maleic Acid (SMA) copolymer. UV–vis absorbance spectroscopy and dynamic-light scattering indicated the formation of BR monomers solubilized in lipid nanodiscs 10.2 ± 0.7 nm in average diameter. Fluorescence and absorbance spectroscopic techniques were utilized to probe conformational, environmental, and rotational changes associated with the tryptophan residues and the covalently-bound retinal moiety of BR upon entrapment in the silica matrix. We show that the immobilized BR in both membrane environments retained its bound retinal cofactor and the ability of the cofactor to undergo conformational changes upon light illumination necessary for BR’s activity as a proton transporter. For purple membrane fragments, the results indicated that the local pH in the pores around BR after encapsulation was important for its stability at temperatures higher than 50 °C. Under the same buffering conditions, retinal was released from silica-encapsulated BR-PM and BR nanodiscs beginning at 80 °C (without a conformational change) and 50 °C (with a conformational change), respectively, reflecting differences in protein-protein (trimeric vs. monomeric) and protein-lipid interactions.

Styrene maleic acid recovers proteins from mammalian cells and tissues while avoiding significant cell death

Detection of protein biomarkers is an important tool for medical diagnostics, typically exploiting concentration of particular biomarkers or biomarker release from tissues. We sought to establish whether proteins not normally released by living cells can be extracted without harming cells, with a view to extending this into biomarker harvest for medical diagnosis and other applications. Styrene maleic acid (SMA) is a polymer that extracts nanodiscs of biological membranes (containing membrane proteins) from cells. Hitherto it has been used to harvest SMA-lipid-membrane protein particles (SMALP) for biochemical study, by destroying the living cellular specimen. In this study, we applied SMA at low concentration to human primary cardiovascular cells and rat vascular tissue, to ‘biopsy’ cell proteins while avoiding significant reductions in cell viability. SMA at 6.25 parts per million harvested proteins from cells and tissues without causing significant release of cytosolic dye (calcein) or reduction in cell viability at 24 and 72 hours post-SMA (MTT assay). A wide range of proteins were recovered (20–200 kDa) and a number identified by mass spectrometry: this confirmed protein recovery from plasma membrane, intracellular membranes and cell cytosol without associated cell death. These data demonstrate the feasibility of non-lethally sampling proteins from cells, greatly extending our sampling capability, which could yield new physiological and/or pathological biomarkers.

Detergent-free isolation of native red blood cell membrane complexes

Over the past few decades, studies on the red blood cell (RBC) membrane gave rise to increasingly sophisticated although divergent models of its structural organization, since investigations were often performed in denaturing conditions using detergents. To access soluble isolated RBC membrane complexes with the preservation of their interactions and conformations, we decided to apply the recent SMALP (Styrene Maleic Acid Lipid Particles) technology to RBC ghosts. Depending on the ionic strength of buffers in which ghost membranes were resuspended, the isolated proteins within SMALPs could differ on Coomassie–stained gels, but with few changes when compared to ghost membrane SDS lysates. We subsequently produced SMALPs derived from ghosts from two different blood group phenotypes, RhD-positive and RhD-negative, both types of RBC expressing the RhCE proteins but only RhD-positive cells being able to express the RhD proteins. This allowed the isolation, by size exclusion chromatography (SEC), of soluble fractions containing the Rh complex, including the RhD protein or not, within SMALPs. The use a conformation-dependent anti-RhD antibody in immunoprecipitation studies performed on SEC fractions of SMALPs containing Rh proteins clearly demonstrated that the RhD protein, which was only present in SMALPs prepared from RhD-positive RBC ghosts, has preserved at least one important conformational RhD epitope. This approach opens new perspectives in the field of the erythroid membrane study, such as visualization of RBC membrane complexes in native conditions by cryo-electron microscopy (CryoEM) or immuno-tests with conformation-dependent antibodies against blood group antigens on separated and characterized SMALPs containing RBC membrane proteins.

Factors influencing the solubilization of membrane proteins from Escherichia coli membranes by styrene–maleic acid copolymers

Styrene–maleic acid (SMA) copolymers are a promising alternative to detergents for the solubilization of membrane proteins. Here we employ Escherichia coli membranes containing KcsA as a model protein to investigate the influence of different environmental conditions on SMA solubilization efficiency. We show that SMA concentration, temperature, incubation time, ionic strength, presence of divalent cations and pH all influence the amount of protein that is extracted by SMA. The observed effects are consistent with observations from lipid-only model membrane systems, with the exception of the effect of pH. Increasing pH from 7 to 9 was found to result in an increase of the solubilization yield of E. coli membranes, whereas in lipid-only model systems it decreased over the same pH range, based on optical density (OD) measurements. Similar opposite pH-dependent effects were observed in OD experiments comparing solubilization of native yeast membranes and yeast lipid-only membranes. We propose a model in which pH-dependent electrostatic interactions affect binding of the polymers to extramembraneous parts of membrane proteins, which in turn affects the availability of polymer for membrane solubilization. This model is supported by the observations that a similar pH-dependence as for SMA is observed for the anionic detergent SDS, but not for the nonionic detergent DDM and that the pH-dependence can be largely overcome by increasing the SMA concentration. The results are useful as guidelines to derive optimal conditions for solubilization of biological membranes by SMA.

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR.

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

Synthesis and evaluation of styrene-maleic acid copolymer conjugated amphotericin B.

Amphotericin B (AmB), which plays a central role in the treatment of systemic fungal infections, is difficult to formulate because it's sparingly soluble in water and organic solvents. We previously prepared AmB-loaded micelles using styrene-maleic acid copolymer (SMA). Although solubilization was achieved by this formulation, stability in the blood circulation was as insufficient as that of Fungizone®, which is a conventional formulation of AmB. Meanwhile, it is well known that polymer-drug conjugates are more stable in circulation than drug-loaded micelles. Therefore, in this study, we developed covalently conjugated SMA-AmB (SMA-AmB conjugate). The SMA-AmB conjugate was found to be soluble and present as micelles in aqueous solution. Furthermore, it was revealed that this micelle behaves as a larger molecule by forming a complex with albumin. The circulation in the blood increased significantly compared to that of Fungizone®, which was suggested to be due to this complex-forming ability. Although in vitro and in vivo antifungal activity of the SMA-AmB conjugate against Saccharomyces cerevisiae was reduced by 1/3 compared to that of Fungizone®, hemolysis decreased to 1/40 or less, and the LD50 decreased to 1/10. In conclusion, it is expected that the SMA-AmB conjugate can be a polymer-therapeutic agent with high antifungal selectivity.

Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of \u03b2-arrestin 2 coupling

Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

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 β-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 β-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.

Covalent functionalized magnetic nanoparticles for crude oil recovery

The purpose of this work is to prepare and characterize new materials based on nanoparticles (NPs) with potential application in the removal of oils.For this purpose, magnetic nanoparticles (MNPs) were covalent functionalized with a styrene-maleic acid copolymer through superficial silanization (MSM), and its adsorption properties were explored and compared to MNPs from crude oil and from brine oil solutions. Magnetic properties enhance recovering and reuse of nanoparticles.As results of these experiments, we found that MSM have higher capacity than MNPs for oil removal from solutions (95% of efficiency at a 0.1 g MSM/L concentration), and more than 90% of the sorption capacity remained after 5 successive sorption-washing cycles, suggesting an excellent reusability. MSM nanofluids were more effective than MNPS for oils displacement in capillary, and it was attributed to MSM tensioactive properties. This behavior was confirmed by the measurement of contact angle for an oil drop in contact with the corresponding nanofluid. MSM were examined also as a crude oil collector using magnetic fields.The reported results show promising data for the separation of the petroleum crude oil from aqueous solution in environmental pollution cleanup.

SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.

Self-Assembly of Polymer-Encased Lipid Nanodiscs and Membrane Protein Reconstitution.

The absence of detergent and curvature makes nanodiscs excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid nanodiscs limits their use in the study of the structure, dynamics, and functions of membrane proteins. In this study, we parameterized and optimized the coarse-graining (CG) bead mapping for two differently charged and functionalized copolymers, containing styrene-maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed nanodisc formation (<8 nm diameter) on a time scale of tens of microseconds using molecular dynamics (MD) simulations. Structural models of ∼2.0 or 4.8 kDa PMAQA and ∼2.2 kDa SMAEA polymer-based lipid nanodiscs highlight the importance of the polymer chemical structure, size, and polymer-lipid ratio in the optimization of the nanodisc structure. The ideal spatial arrangement of polymers in nanodiscs, nanodisc size, and thermal stability obtained from our MD simulation correlates well with the experimental observations. The polymer-nanodiscs were tested for the reconstitution of single-pass or multipass transmembrane proteins. We expect this study to be useful in the development of novel polymer-based lipid nanodiscs and for the structural studies of membrane proteins.

Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs.

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

From polymer chemistry to structural biology: The development of SMA and related amphipathic polymers for membrane protein extraction and solubilisation.

Nanoparticles assembled with poly(styrene-maleic acid) copolymers, identified in the literature as Lipodisq, SMALPs or Native Nanodisc, are routinely used as membrane mimetics to stabilise protein structures in their native conformation. To date, transmembrane proteins of varying complexity (up to 8 beta strands or 48 alpha helices) and of a range of molecular weights (from 27 kDa up to 500 kDa) have been incorporated into this particle system for structural and functional studies. SMA and related amphipathic polymers have become versatile components of the biochemist's tool kit for the stabilisation, extraction and structural characterization of membrane proteins by techniques including cryo-EM and X-ray crystallography. Lipodisq formation does not require the use of conventional detergents and thus avoids their associated detrimental consequences. Here the development of this technology, from its fundamental concept and design to the diverse range of experimental methodologies to which it can now be applied, will be reviewed.

Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.

Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

Influence of Mg2+ and Ca2+ on nanodisc formation by diisobutylene/maleic acid (DIBMA) copolymer.

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

Structural characterization of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using EPR spectroscopy.

Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing homogenous and functional lipid membrane mimetic systems into which membrane proteins can properly fold and function. It has recently been shown that styrene-maleic acid (SMA) copolymers act as a macromolecular surfactant and therefore facilitate the formation of disk-shaped lipid bilayer nanoparticles (styrene-maleic acid copolymer-lipid nanoparticles (SMALPs)) that retain structural characteristics of native lipid membranes. We have previously reported controlled synthesis of SMA block copolymers using reversible addition-fragmentation chain transfer (RAFT) polymerization, and that alteration of the weight ratio of styrene to maleic acid affects nanoparticle size. RAFT-synthesis offers superior control over SMA polymer architecture compared to conventional radical polymerization techniques used for commercially available SMA. However, the interactions between the lipid bilayer and the solubilized RAFT-synthesized SMA polymer are currently not fully understood. In this study, EPR spectroscopy was used to detect the perturbation on the acyl chain upon introduction of the RAFT-synthesized SMA polymer by attaching PC-based nitroxide spin labels to the 5th, 12th, and 16th positions along the acyl chain of the lipid bilayer. EPR spectra showed high rigidity at the 12th position compared to the other two regions, displaying similar qualities to commercially available polymers synthesized via conventional methods. In addition, central EPR linewidths and correlation time data were obtained that are consistent with previous findings.

Nanodiscs bounded by styrene-maleic acid allow trans-cis isomerization of enclosed photoswitches of azobenzene labeled lipids.

Styrene-and-maleic acid (SMA) copolymers behave as amphipathic belts encircling lipids in the form of nanodiscs. It is unclear to what extent the SMA belt affects the order and dynamics of the enclosed lipids. We aimed to obtain insight into this by making use of synthetic azobenzene-labeled phospholipids incorporated into di-16:0 PC nanodiscs. Azobenzene lipids undergo geometric isomerization upon exposure to light at 365 nm, resulting in the formation of cis-isomers that possess a larger cross-sectional area than the trans-isomers. The influence of the lipid properties on the kinetics and extent of isomerization of the azobenzene groups was first tested in large unilamellar vesicles constituted by lipid mixtures with different packing properties of the acyl chains. Fastest isomerization kinetics were found when azolipids were present in membranes supplemented with lysolipids and slowest in those supplemented with di-unsaturated lipids, suggesting that the isomerization rate is sensitive to the lateral pressure profile in the lipid bilayer and hence may be considered a convenient tool to monitor packing properties of lipids enclosed in nanodiscs. When azolipids were incorporated in SMA-bounded nanodiscs, azolipid isomerization was found to take place readily, indicating that SMA polymers behave as rather flexible belts and allow expansion of the enclosed lipid material.

Synthesis and In vitro/In vivo Characterization of Raloxifene Grafted Poly(Styrene Maleic Acid)-Poly(Amide-Ether-Ester-Imide) Micelles for Targeted Delivery of Docetaxel in G Protein-Coupled Estrogen Receptor Breast Cancer.

To reduce the nonspecifically distribution of chemotherapeutic agents throughout the whole body, which causes severe toxicity in normal tissues, targeting them towards a receptor overexpressed on tumor tissue, is a promising method for cancer therapy. The aim of the present study was development of novel copolymeric micelles of raloxifene targeted Styrene Maleic Acid-Poly Amide Ether Ester Imide-Poly Ethylene Glycol (SMA-PAEEI-PEG-RA) and loading them with Docetaxel (DTX). Successful synthesis of the targeted copolymer was confirmed by FTIR and C-NMR spectroscopy. The micelles physicochemical properties like morphology, particle size, poly dispersity index, zeta potential, drug loading, release, stability, in vitro cytotoxicity and cellular uptake were analyzed. The in vivo antitumor activity of DTX-loaded micelles were assessed and compared with free DTX and non-targeted micelles in breast cancer bearing Balb-c mice. Particle sizes, zeta potentials and the encapsulation efficiency of the drug in targeted micelles were 115.9- 142.8 nm, -4.9 to -12.9 mV, and 54.1-67.8%, respectively. Cell toxicity tests showed that IC50 of DTX-loaded SMAPAEEI- PEG-RA micelles increased five-fold as compared with free DTX. Survival rate of the mice improved more effectively than free DTX so that, the percentage of increase in lifespan (ILS%) and the tumor inhibition ratio (TIR) changed from 41.66% and 51.19% in free drug to 83.33% and 78.57% in the targeted micelles, respectively. Therefore, the raloxifene conjugated PEG-derived micelles may provide a novel and effective delivery system for DTX in breast cancer.

Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins.