Styrene-maleic Acid Research Articles

Alternatives to Styrene- and Diisobutylene-Based Copolymers for Membrane Protein Solubilization via Nanodisc Formation.

Styrene-maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native-like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α-olefin-maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene-co-maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I-containing nanodiscs that retain an annulus of native lipids and a native-like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine-tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.

Sulfonated polystyrenes: pH and Mg2+-insensitive amphiphilic copolymers for detergent-free membrane protein isolation

Amphiphilic polymers are increasingly applied in the detergent-free isolation and functional studies of membrane proteins. However, the carboxylate group present in the structure of many popular variants, such as styrene-maleic acid (SMA) copolymers, brings limitations in terms of polymer sensitivity to precipitation at acidic pH or in the presence of divalent metal cations. Herein, we addressed this problem by replacing carboxylate with the more acidic sulfonate groups. To this end, we synthesized a library of amphiphilic poly[styrene-co-(sodium 4-styrene sulfonate)] copolymers (termed SSS), differing in their molecular weight and overall polarity. Using model cell membranes (Jurkat), we identified two copolymer compositions (SSS-L30 and SSS-L36) that solubilized membranes to an extent similar to SMA. Interestingly, the density gradient ultracentrifugation/SDS-PAGE/Western blotting analysis of cell lysates revealed a distribution of studied membrane proteins in the gradient fractions that was different than for SMA-solubilized membranes. Importantly, unlike SMA, the SSS copolymers remained soluble at low pH and in the presence of Mg2+ ions. Additionally, the solubilization of DMPC liposomes by the lead materials was studied by turbidimetry, DLS, SEC, and high-resolution NMR, revealing, for SSS-L36, the formation of stable particles (nanodiscs), facilitated by the direct hydrophobic interaction of the copolymer phenyls with lipid acyl chains.

Label-free measurement of antimicrobial peptide interactions with lipid vesicles and nanodiscs using microscale thermophoresis

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (KD) and the partitioning coefficient between the aqueous- and membrane phases (KP) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of KD by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured KD values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs’ minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions or mapping of sample conditions in preparation for more advanced studies that rely on expensive sample preparation, labelling and/or instrument time.

Electron paramagnetic resonance spectroscopic characterization of the human KCNE3 protein in lipodisq nanoparticles for structural dynamics of membrane proteins

One of the major challenges in solubilization of membrane proteins is to find the optimal physiological environment for their biophysical studies. EPR spectroscopy is a powerful biophysical technique for studying the structural and dynamic properties of macromolecules. However, the challenges in the membrane protein sample preparation and flexible motion of the spin label limit the utilization of EPR spectroscopy to a majority of membrane protein systems in a physiological membrane-bound state. Recently, lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. However, its detail characterization for membrane protein studies is still poorly understood. Recently, we characterized the potassium channel membrane protein KCNQ1 voltage sensing domain (KCNQ1-VSD) and KCNE1 reconstituted into lipodisq nanoparticles using EPR spectroscopy. In this study, the potassium channel accessory protein KCNE3 containing flexible N- and C-termini was encapsulated into proteoliposomes and lipodisq nanoparticles and characterized for studying its structural and dynamic properties using nitroxide based site-directed spin labeling EPR spectroscopy. CW-EPR lineshape analysis data indicated an increase in spectral line broadenings with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated an enhanced quality of distance measurements with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles, when compared to proteoliposomes. These results agree with the solution NMR structural structure of the KCNE3 and EPR studies of other membrane proteins in lipodisq nanoparticles. This study along with our earlier studies will provide the reference characterization data that will provide benefit to the membrane protein researchers for studying structural dynamics of challenging membrane proteins.

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening.

Membrane protein (MP)-based biomaterials have a wide range of applications in drug screening, antigen detection, and ligand-receptor interaction analysis. Traditional MP immobilization methods have the disadvantage of disordered protein immobilization orientation, leading to the shielded binding domain and unreliable binding pattern. Herein, we describe a site-specific covalent immobilization of MPs, which utilizes the styrene maleic acid (SMA) detergent-free extraction method of MPs as well as the covalent reaction between His-tag and divinyl sulfone (DVS). As an example, we covalently immobilized angiotensin-converting enzyme 2 (ACE2) on a cell membrane chromatography system (ACE2-His-SMALPs/CMC) in a site-specific manner and verified the specificity and stability of this system. This technique significantly improves the service life compared to the physisorption CMC column. The improved protein immobilization strategies of the ACE2-His-SMALPs/CMC system enable it to effectively recognize SARS-CoV-2 pseudoviral particles as well as detect viral particles in ambient air once combined with an aerosol collector; as a powerful ligand biosensor, the ACE2-His-SMALPs/CMC system was used to screen for compounds with anti-SARS-CoV-2 pseudovirus activity. In conclusion, the optimized MP immobilization strategy has been successfully applied to CMC technology, showing enhanced stability and sensitivity, which can provide an efficient and convenient membrane protein immobilization method for biomaterials.

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.

Asymmetric conformations of cleaved HIV-1 envelope glycoprotein trimers in styrene-maleic acid lipid nanoparticles

During virus entry, the pretriggered human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer initially transits into a default intermediate state (DIS) that remains structurally uncharacterized. Here, we present cryo-EM structures at near-atomic resolution of two cleaved full-length HIV-1 Env trimers purified from cell membranes in styrene-maleic acid lipid nanoparticles without antibodies or receptors. The cleaved Env trimers exhibited tighter subunit packing than uncleaved trimers. Cleaved and uncleaved Env trimers assumed remarkably consistent yet distinct asymmetric conformations, with one smaller and two larger opening angles. Breaking conformational symmetry is allosterically coupled with dynamic helical transformations of the gp41 N-terminal heptad repeat (HR1N) regions in two protomers and with trimer tilting in the membrane. The broken symmetry of the DIS potentially assists Env binding to two CD4 receptors—while resisting antibody binding—and promotes extension of the gp41 HR1 helical coiled-coil, which relocates the fusion peptide closer to the target cell membrane.

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.

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

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

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

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

Cryo-EM structure of the four-subunit Rhodobacter sphaeroides cytochrome bc1 complex in styrene maleic acid nanodiscs

Cytochrome bc1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b, cytochrome c1, and the Rieske iron-sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 complex from the purple phototrophic bacterium Rhodobactersphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene-maleic acid copolymer to purify the R. sphaeroides cytochrome bc1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c1 subunits. We observe a quinone at the Qo quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.

Composition of raft-like cell membrane microdomains resistant to styrene-maleic acid copolymer (SMA) solubilization

An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).

Structural Insights into Polymer-Bounded Lipid Nanodiscs.

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

Nanodisc scaffold alters the structure of a pentameric ligand-gated ion channel revealing new conformations.

Lipid nanodiscs are a powerful tool for structural and biochemical characterization of membrane proteins including ion channels. Many distinct nanodisc scaffolds are widely used, but how the nanodisc affects the structure of ion channels is poorly understood. Using single particle cryo-EM, we determined apo and agonist-bound structures of the pentameric ligand-gated ion channel (pLGIC), ELIC, in different nanodiscs: MSP1E3D1, SMA (styrene maleic acid), saposin, and spMSP1D1 (a circularized nanodisc). All structures used the same lipids (2:1:1 POPC:POPE:POPG) for reconstitution; in this lipid environment, ELIC exhibits robust responses to agonist. Strikingly, each nanodisc scaffold produced a distinct structure. In the extracellular domain (ECD) and the ECD-TMD (transmembrane domain) interface, the agonist-bound structures show incremental and concerted conformational differences associated with activation (least to most activated: SMA > MSP1E3D1 > saposin > spMSP1D1). The same trend is observed in the TMD where more activated stuctures show an outward expansion of the TMD helices. While agonist-bound structures in SMA, MSP1E3D1 and saposin show a pore with constrictions at 16’ and 9’ consistent with pre-active conformations, the structure in the circularized nanodisc, spMSP1D1, shows opening at 16’ and a constriction at 2’ consistent with a desensitized conformation. Interestingly, both apo and agonist-bound structures of ELIC in MSP1E3D1 show a uniquely contracted ECD with limited activation in the agonist-bound structure. Reconstitution of ELIC in different nanodisc scaffolds appears to produce different nanodisc diameters with the smallest being MSP1E3D1, followed by saposin, and spMSP1D1. Therefore, larger nanodiscs are associated with agonist-bound structures with greater activation. Overall, the results indicate that nanodisc scaffold significantly affects the conformation of a pLGIC, and that larger nanodiscs, achieved using a circularized nanodisc scaffold, may better mimic a native membrane environment.

Copolymeric nanodiscs do not retain the native membrane environment, but do maintain protein-lipid interactions.

Polymer stabilized lipid-protein nanoparticles have become invaluable tools in membrane biology since they can isolate individual proteins with their surrounding lipids. These nanoparticles, termed nanodiscs, have historically relied on membrane scaffolding proteins, but can now be formed using amphipathic copolymers that directly intercalate into membranes. Since copolymeric nanodiscs are formed directly from membranes without requiring detergent solubilization, they have been presumed to retain the native membrane environment, but this assumption has not been thoroughly tested. To test if native membrane lipid packing is retained in styrene-maleic acid (SMA) and diisobutylene-maleic acid (DIBMA) stabilized nanodiscs, lipid packing was analyzed using solvatochromic dyes Laurdan, C-Laurdan, and Pro12A. SMA and DIBMA copolymeric nanodiscs were formed from membranes increasing in complexity from single and binary lipid mixture, to biomimetic ternary mixtures composed of palmitoyl-sphingomyelin (PSM), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and cholesterol, and ultimately to mammalian cellular membranes. For all synthetic model membranes, lipid packing of fluid-disordered membranes was significantly increased in both SMA and DIBMA nanodiscs compared to intact membranes, with DIBMA being the least perturbing. In contrast, lipid packing in ordered membranes was unaffected. For cellular membranes, lipid packing was also higher inside nanodiscs than in native membranes. Our data challenges the assumption that native membrane properties are retained inside nanodiscs. While not maintaining native membrane properties, nanodiscs can be useful models for studying protein-lipid interactions, as they are composed of membrane lipids. Interestingly, nanodiscs can exchange lipids through collisions, allowing tuning of their lipid compositions. We used this capability to analyze the preferred surrounding lipidome of multi-pass transmembrane proteins. Altogether, our studies demonstrate that copolymeric nanodiscs have different properties to those of intact membranes, but they are useful models in studying the paralipidomes of membrane proteins.

A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes

From the discovery of the first membrane-interacting polymer, styrene maleic-acid (SMA), there has been a rapid development of membrane solubilising polymers. These new polymers can solubilise membranes under a wide range of conditions and produce varied sizes of nanoparticles, yet there has been a lack of broad comparison between the common polymer types and solubilising conditions. Here, we present a comparative study on the three most common commercial polymers: SMA 3:1, SMA 2:1, and DIBMA. Additionally, this work presents, for the first time, a comparative characterisation of polymethacrylate copolymer (PMA). Absorbance and dynamic light scattering measurements were used to evaluate solubilisation across key buffer conditions in a simple, adaptable assay format that looked at pH, salinity, and divalent cation concentration. Lipid-polymer nanoparticles formed from SMA variants were found to be the most susceptible to buffer effects, with nanoparticles from either zwitterionic DMPC or POPC:POPG (3:1) bilayers only forming in low to moderate salinity (< 600 mM NaCl) and above pH 6. DIBMA-lipid nanoparticles could be formed above a pH of 5 and were stable in up to 4 M NaCl. Similarly, PMA-lipid nanoparticles were stable in all NaCl concentrations tested (up to 4 M) and a broad pH range (3–10). However, for both DIBMA and PMA nanoparticles there is a severe penalty observed for bilayer solubilisation in non-optimal conditions or when using a charged membrane. Additionally, lipid fluidity of the DMPC-polymer nanoparticles was analysed through cw-EPR, showing no cooperative gel-fluid transition as would be expected for native-like lipid membranes.

Investigation into aquaporin 1's conserved structural features and the C-terminal tail.

The integral membrane protein human aquaporin-1 (AQP1), for the most part, has been well characterized structurally. A few X-ray and cryo-EM structures exist, however information regarding several functionally important residues and motifs that are conserved throughout the aquaporin family have not been fully explored. As well, the long, flexible C-terminal tail is not well-characterized, even though evidence suggests it is important in several protein-protein interaction events that govern AQP1's translocation to the cellular membrane, and in turn its activity. These structural features are important in further understanding AQP1's function, as it becomes increasingly more medically relevant and a possible drug target, with its involvement in tumour cell migration and angiogenesis. In this work, we explored the feasibility of probing AQP1's C-terminal tail in solution using NMR spectroscopy and a novel polymer nanodisc platform. The nanodiscs encapsulating AQP1 have taken advantage of the styrene-maleic acid co-polymer to directly solubilize Pichia pastoris membranes. The resulting styrene-maleic acid lipid nanoparticles (SMALPs), suspended freely in solution, then act as a scaffold to study protein-protein interactions on the C-terminal tail of AQP1. Preliminary studies of the AQP1-SMALPs have resulted in well resolved 1H-15N HSQC spectra, and further microscopy and spectroscopy studies have determined the size and relative stability of the discs. Furthermore, we have also investigated several key conserved residues in AQP1, that through prior work, are hypothesized to be heavily involved in overall protein fold stability. Through mutagenesis experiments coupled with H/D exchange and Fourier transform infrared spectroscopy, we were able to monitor the differences in stability given the extent of H/D exchange at multiple temperature points. These experiments will yield unique structural insights into AQP1, and present a foundation towards discovering new protein partner interactions.

Lipid packing is disrupted in copolymeric nanodiscs compared with intact membranes

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

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.