Styrene-maleic Acid Copolymer Research Articles

Purification and catalysis of choline dehydrogenase from Escherichia coli

Choline dehydrogenase (CHDH) is a membrane-bound enzyme belonging to the glucose-methanol-choline (GMC) oxidoreductase superfamily, which is characterized by a crucial FAD-binding domain essential for catalytic function. CHDH catalyzes the oxidation of choline to betaine aldehyde, which is further oxidized to betaine, a vital osmoprotectant and methyl donor for cellular physiology and metabolism. However, the detailed catalytic mechanism of CHDH still remains poorly understood. In our investigation, we gained purity E. coli CHDH samples in DDM (n-dodecyl-β-D-maltoside) and SMA (styrene maleic acid) copolymer respectively and examined their structural composition and catalytic activity separately. Our findings demonstrated the effectiveness of SMA, commonly employed for extracting transmembrane proteins and can preserve the natural bio-membrane environment surrounding the enzyme, in extracting peripheral membrane proteins like CHDH here, which lacks transmembrane helices. CHDH exhibited a trimeric conformation in SMA, whereas it existed as monomers in DDM, as determined by our negative staining analysis. Our experiments also revealed that highly pure E. coli CHDH could only oxidize choline to betaine aldehyde but failed to further oxidize betaine aldehyde to betaine as determined by the biochemical and enzymatic reaction kinetic assays. In addition, the enzyme in SMA displayed greater catalytic activity compared to that in DDM. Furthermore, we confirmed the crucial role of His473, which is hypothesized to be a critical site for substrate binding from our structural comparative analysis between CHDH and its highly homologous choline oxidase, in the catalytic activity of the enzyme through gene mutation. Our work also sheds light on CHDH’s contribution to cellular osmotic tolerance through gene knockout. This research enhances our better understanding of CHDH within cellular biochemistry and metabolic pathways.

Esterified styrene-maleic acid copolymer modified silica as mixed-mode polymer-brush stationary phases for chromatographic separation

Styrene-maleic acid (SMA) copolymer has received much attention for its excellent solubilization characteristics. In this work, SMA copolymer brush-based chromatographic stationary phases were exploited and developed for the first time. First, SMA copolymer brush was in situ grown on the surface of spherical silica via living/controlled reversible addition-fragmentation chain transfer (RAFT) polymerization method. Subsequently, as a proof-of-concept demonstration, the copolymer was esterified by diethylene glycol mono-2-ethylhexyl ether (DGME) and 2-(2-ethylhexyloxy) ethanol (EHOE), respectively. The obtained Sil-SMA-DGME and Sil-SMA-EHOE copolymer-brush chromatographic stationary phases were characterized by transmission electron microscopy, Fourier transform infrared spectrometer, X-ray photoelectron spectroscopy, and thermogravimetric analysis, respectively. The chromatographic retention mechanism indicated that both the two packed columns exhibited hydrophilic/reverse mixed-mode retention modes. The maximum column efficiency was up to 71,000 N/m. The chromatographic separation performance evaluation indicated that the novel kind of stationary phases had excellent separation capabilities for hydrophilic, hydrophobic compounds and phospholipid standards. In addition, by combination with mass spectrometry identification, the Sil-SMA-DGME column was further exploited for separation and identification of phospholipids in human lung cancer cells. Totally, 9 classes including 186 phospholipid species were successfully identified. The results demonstrated the promising application prospects of the novel kind of SMA copolymer-brush chromatographic stationary phases.

Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, i.e. (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

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

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

Conformational transitions of maleic acid segment drive pH induced changes in SMA polymer structure and solubility

The popularity of styrene-maleic acid (SMA) copolymers is based on their ability to solubilize biological membranes into nanoscale disks facilitating the extraction and purification of membrane proteins. Unfortunately, these disks are unstable in acidic environments which is due to the conformational transition of the SMA polymer. In this work, using a combination of NMR, IR and fluorescence spectroscopy together with DLS and quantum chemical calculations, we have investigated the pH-mediated conformational transition in the SMA polymer. With decreasing pH, polymer undergoes the following changes: 1) the dihedral angle in the maleic moieties of the polymer changes from 180° to 60° and then reaches 50°; 2) the carboxyl groups of the maleic fragment form hydrogen bonds with other maleic fragments, leading to aggregation of the SMA molecules; 3) the phenyl rings of the styrene fragments are brought closer together and the hydrophobicity of the environment inside the SMA polymer globule increases.

Solubilization of Oligomeric Cell-Free Synthesized Proteins Using SMA Copolymers.

Although membrane proteins are abundant in nature, their investigation is limited due to bottlenecks in heterologous overexpression and consequently restricted accessibility for downstream applications. In this chapter, we address these challenges by presenting a fast and straightforward synthesis platform based on eukaryotic cell-free protein synthesis (CFPS) and an efficient solubilization strategy using styrene-maleic acid (SMA) copolymers. We demonstrate CFPS of TWIK-1, a dimeric ion channel, based on Sf21 (Spodoptera frugiperda) insect lysate showing homooligomerization and N-glycosylation enabled by endoplasmic reticulum-derived microsomes. Furthermore, we employ SMA copolymers for protein solubilization, which preservesthe native-like microsomal environment. This approach not only retains the solubilized protein's suitability for downstream applications but also maintains the oligomerization and glycosylation of TWIK-1 post-solubilization. We validate the solubilization procedure using autoradiography, particle size analysis, and biomolecular fluorescence assay and confirm the very efficient, structurally intact solubilization of cell-free synthesized TWIK-1.

Single-Molecule Imaging of Ion Channels Isolated in Detergent and Styrene-Maleic Acid Copolymer

Single-Molecule Imaging of Ion Channels Isolated in Detergent and Styrene-Maleic Acid Copolymer

Preparation and in vitro evaluation of amphotericin B-loaded nano-formulations for both intravenous and ophthalmic administration using 3-aminophenylboronic acid-conjugated styrene-maleic acid copolymers

Amphotericin B (AmB) is a widely used antifungal drug. However, its insolubility in water and high toxicity pose challenges to its clinical application. The aim of this study was to improve the solubility, to reduce toxicity, and to confer albumin- and mucin-binding properties. Therefore, we developed an AmB nano-formulation (APB-SMA/AmB) using a styrene-maleic acid copolymer modified with 3-aminophenylboronic acid (APB-SMA). APB-SMA/AmB was characterized and evaluated for drug content, particle size, toxicity, antifungal activity, and albumin- and mucin-binding properties.APB-SMA contains APB molecules bound to styrene-maleic acid copolymer (SMA), which can form complexes with diols. APB-SMA/AmB exhibits a small and a large peak at approximately 330 nm and 410 nm respectively, in the UV–visible spectrum, indicating the presence of a large amount of monomeric AmB, which is less toxic than its oligomeric form. The particle size of the formulations was in the nanoscale range. APB-SMA/AmB showed reduced hemolytic activity compared with AmB and the other formulations. The antifungal activity of APB-SMA/AmB is derived from AmB, and a higher APB-SMA content in the formulation improves the therapeutic index. Furthermore, APB-SMA/AmB demonstrated in vitro albumin- and mucin-binding properties, suggesting its potential as a drug delivery system for improving in vivo blood circulation and corneal retention. In conclusion, APB-SMA/AmB is an effective water-soluble nano-formulation for fungal infections and may be applicable in both injectable and ophthalmic formulations.

Uptake Pathway of Styrene Maleic Acid Copolymer-Coated Lipid Emulsions Under Acidic Tumor Microenvironment

The purpose of this study was to elucidate and compare styrene maleic acid copolymer (SMA)-coated lipid emulsions (SMA emulsions) uptake pathway in vascular endothelial cells and surrounding cancer cells under not only neutral but also acidic pH, which is often observed in tumor microenvironment. DiI-labeled SMA emulsions were prepared using 1-palmitoyl-2-oleoyl-sn‑glycero-3-phosphocholine and triolein. In murine melanoma B16-BL6 (B16) cells and human umbilical vein endothelial cells (HUVEC), DiI-labeled SMA emulsions uptake under near-neutral (pH 7.4) and acidic (pH 6.0) conditions was determined by fluorescent analysis. SMA emulsions were taken up more efficiently into HUVEC than B16 cells under acidic condition in a temperature-dependent manner. Uptake study using endocytosis inhibitors showed that SMA emulsions were taken up by macropinocytosis and clathrin-mediated endocytosis in B16 cells. In HUVEC, however, they were taken up by clathrin- and caveolae-independent, but dynamin-dependent pathway. SMA emulsions would be internalized efficiently into vascular endothelial cells as well as cancer cells under acidic microenvironment via different endocytosis pathways. SMA emulsions could be a promising drug delivery carrier for anti-angiogenic drugs.

Exploring the therapeutic potential of a nano micelle containing a carbon monoxide-releasing molecule for metabolic-associated fatty liver disease by modulating hypoxia-inducible factor-1α.

Metabolic-associated fatty liver disease (MAFLD) encompasses a spectrum of chronic liver diseases, including steatohepatitis, cirrhosis, and liver cancer. Despite the increasing prevalence and severity of MAFLD, no approved pharmacological interventions are currently available. Hypoxia-inducible factor-1α (HIF-1α) has emerged as a crucial early mediator in the pathogenesis of MAFLD. Previously, we demonstrated the potent anti-inflammatory properties of the nano-designed carbon monoxide (CO) donor, styrene maleic acid copolymer (SMA) encapsulating CO-releasing molecule (SMA/CORM2), which effectively suppressed HIF-1α in various inflammatory disorders. Here, we investigated the therapeutic potential of SMA/CORM2 in a mouse model of MAFLD induced by a high-fat methionine- and choline-deficient (HF-MCD) diet. Following 4 weeks of HF-MCD diet consumption, we observed pronounced hepatic lipid accumulation accompanied by disrupted lipid metabolism, polarization of macrophages towards the pro-inflammatory M1 phenotype, activation of the NLRP3 inflammasome, and upregulation of the TGF-β fibrosis signaling pathway. Notably, the early and upstream event driving these pathological changes was the upregulation of HIF-1α. Treatment with SMA/CORM2 (10mg/kg, three times per week) led to a significant increase in CO levels in both the circulation and liver, resulting in remarkable suppression of HIF-1α expression even before the onset of apparent pathological changes induced by the HF-MCD diet. Consequently, SMA/CORM2 administration exerted a significantly protective and therapeutic effect on MAFLD. In vitro studies using hepatocytes treated with high concentrations of fatty acids further supported these findings, as knockdown of HIF-1α using short hairpin RNA (shRNA) elicited similar effects to SMA/CORM2 treatment. Collectively, our results highlight the therapeutic potential of SMA/CORM2 in the management of MAFLD through suppression of HIF-1α. We anticipate that SMA/CORM2, with its ability to modulate HIF-1α expression, may hold promise for future applications in the treatment of MAFLD. STATEMENT OF SIGNIFICANCE: Carbon monoxide (CO) is a crucial gaseous signaling molecule that plays a vital role in maintaining homeostasis and is a potential target for treating many inflammatory diseases. Developing drug delivery systems that can deliver CO stably and target specific tissues is of great interest. Our team previously developed a nano micellar CO donor, SMA/CORM2, which exhibits superior bioavailability to native CORM2 and shows therapeutic potential in many inflammatory disease models. In this study, we showed that SMA/CORM2, through controlled CO release, significantly ameliorated steatohepatitis and liver fibrosis induced by an HF-MCD diet by suppressing an HIF-1α mediated inflammatory cascade. These findings provide new insight into the anti-inflammatory function of CO and a promising approach for controlling metabolic-associated fatty liver disease.

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.

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.

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).

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.

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.

Highly differentiated multi-stimuli-responsive fluorescence performance of tetraphenylethylene-containing styrene–maleic acid copolymers induced by macromolecular architecture control

TPE-containing styrene–maleic acid copolymers with a diverse chain architecture display highly differentiated fluorescence responsiveness to multiple stimuli.