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Traditional treatments for head and neck squamous cell carcinoma (HNSCC) such as surgery, radiation therapy, and chemotherapy, often have severe side effects. Local delivery of chemotherapeutic agents can be a promising approach to minimise systemic toxicity and improve efficacy. Lauric acid (LA), was explored as a novel injectable thermosensitive drug reservoir as a depot for sustained release of anticancer drugs to treat HNSCC. LA was characterised in terms of melting temperature and gelation time. The efficacy of LA-based drug formulations was tested in vitro in a HNSCC cell line and in vivo in a mouse model of HNSCC. LA was modified to have a melting point of 38.5 °C and a gelation time of 40 s at 37.5 °C, rendering it suitable for injection at body temperature. LA- based doxorubicin (DOXO) formulation showed slow release with a maximum of 18% release after 3 days. The in vitro study showed that LA enhanced the cytotoxic effect of DOXO. LA combined with DOXO prevented tumour progression and LA alone significantly reduced the original tumour volume compared to the untreated control group. These findings confirmed that LA can function as practical carrier for the local delivery of chemotherapeutics and provides a safe and simple strategy for the delivery of hydrophobic anticancer drugs and warrant further testing in clinical trials. [ABSTRACT FROM AUTHOR]

Approaches aiming to recover proteins without denaturation represent attractive strategies. To accomplish this, a membrane lysis agent based on poly(styrene-alt-maleic acid) or PSMA was synthesized by photopolymerization using Irgacure® 2959 and carbon tetrabromide (CBr4) as a radical initiator and a reversible chain transfer agent, respectively. Structural elucidation of our in-house synthesized PSMA, so-called photo-PSMA, was performed by using NMR spectroscopy. The use of this photo-PSMA in soybean enzyme extraction was also demonstrated for the first time in this study. Without a severe cell rupture, energy input or any organic solvent, recovery of lipolytic enzymes directly into nanometric-sized particles was accomplished in one-step process. Due to the improved structural regularity along the photo-PSMA backbone, the most effective protective reservoir for enzyme immobilization was generated through the PSMA aggregation. Formation of such reservoir enabled soybean enzymes to be shielded from the surroundings and resolved in their full functioning state. This was convinced by the increased specific lipolytic activity to 1,950 mU/mg, significantly higher than those of sodium dodecyl sulfate (SDS) and the two commercially-available PSMA sources (1000P and 2000P). Our photo-PSMA had thus demonstrated its great potential for cell lyse application, especially for soybean hydrolase extraction.

AbstractBee propolis has been used in alternative medicine to treat various diseases. Due to its limited water solubility, it is often used in combination with alcohol solvents, causing skin irritation and immune response. To solve this, the new drug delivery system, based on the lipid nanodiscs of 1,2-dimyristoyl-sn-glycero-3-phosphochline (DMPC) and poly(styrene-alt-maleic acid) (PSMA), were created in an aqueous media. At the excess polymer concentrations, the PSMA/DMPC complexation produced the very fine nanoparticles (18 nm). With the increased molar ratio of styrene to maleic acid (St/MA) in the copolymer structure, the lipid nanodisc showed the improved encapsulation efficiency (EE%), comparing to their corresponding aqueous formulations. The maximum value had reached to around 20% when using the 2:1 PSMA precursor. Based on the cytotoxicity test, these nanoparticles were considered to be non-toxic over the low dose administration region (

Plant antioxidants can be applied in the management of various human diseases. Despite these, extraction of these compounds still suffers from residual solvent impurities, low recovery yields, and the risks of undesirable chemical changes. Inspired by the protein–lipid interactions in the cell membranes, we proposed using poly(styrene-alt-maleic acid) (PSMA) to destabilize and associate with the bilayer lipids into the membrane-like nanodiscs. Such nanostructures could serve as protective reservoirs for the active compounds to reside with preserved bioactivities. This concept was demonstrated in the antioxidant extraction from robusta coffee leaves. Results indicated that aqueous PSMA extraction (no buffer agent) yielded products with the highest contents of phenolic acids (11.6 mg GAE·g−1) and flavonoids (9.6 mg CE·g−1). They also showed the highest antioxidant activity (IC50 = 3.7 µg·mL−1) compared to those obtained by typical sodium dodecyl sulfate and water extraction. This biomimetic approach could be considered for developing environmentally friendly extraction protocols in the future.

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification. SMALPs were immobilized on a surface plasmon resonance biosensor chip to establish a P-gp inhibitor screening system and calculated the affinity between P-gp and ligands. [Display omitted] [ABSTRACT FROM AUTHOR]

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

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

Approaches aiming to recover proteins without denaturation represent attractive strategies. To accomplish this, a membrane lysis agent based on poly(styrene- alt -maleic acid) or PSMA was synthesized by photopolymerization using Irgacure® 2959 and carbon tetrabromide (CBr 4 ) as a radical initiator and a reversible chain transfer agent, respectively. Structural elucidation of our in-house synthesized PSMA, so-called photo-PSMA, was performed by using NMR spectroscopy. The use of this photo-PSMA in soybean enzyme extraction was also demonstrated for the first time in this study. Without a severe cell rupture, energy input or any organic solvent, recovery of lipolytic enzymes directly into nanometric-sized particles was accomplished in one-step process. Due to the improved structural regularity along the photo-PSMA backbone, the most effective protective reservoir for enzyme immobilization was generated through the PSMA aggregation. Formation of such reservoir enabled soybean enzymes to be shielded from the surroundings and resolved in their full functioning state. This was convinced by the increased specific lipolytic activity to 1,950 mU/mg, significantly higher than those of sodium dodecyl sulfate (SDS) and the two commercially-available PSMA sources (1000P and 2000P). Our photo-PSMA had thus demonstrated its great potential for cell lyse application, especially for soybean hydrolase extraction., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water-soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques. Nuclear magnetic resonance is one such high-resolution approach that looks at the structure and dynamics of a protein with atomic level precision. Recently, there has been a breakthrough in making nanodiscs more susceptible for structure determination by solution NMR, yet it still remains to become the preferred choice for a membrane mimetic. In this practical review, we provide a general discourse on nanodisc and its application to solution NMR. We also offer potential solutions to remediate the technical challenges associated with nanodisc preparation and the choice of proper experimental set-ups. Along with discussing several structural applications, we demonstrate an alternative use of nanodiscs for functional studies, where we investigated the phosphorylation of a cell surface receptor, integrin. This is the first successful manifestation of observing activated receptor phosphorylation in nanodisc through NMR. We additionally present an on-column method for nanodisc preparation with multiple strategies and discuss the potential use of alternative nanoscale phospholipid bilayer systems like styrene maleic acid lipid disc and saposin-A lipoprotein disc.

Featured Application: Nanodiscs formed by styrene maleic acid copolymers allow separation of biologically intact protein complexes with native ligands for syst-OMICS analysis. The omics disciplines remain largely distinct sciences due to the necessity of separating molecular classes for different assays. For example, water-soluble and lipid bilayer-bound proteins and metabolites are usually studied separately. Nonetheless, it is at the interface between these sciences where biology happens. That is, lipid-interacting proteins typically recognize and transduce signals and regulate the flow of metabolites in the cell. Technologies are emerging to converge the omics. It is now possible to separate intact membrane:protein assemblies (memteins) directly from intact cells or cell membranes. Such complexes mediate complete metabolon, receptor, channel, and transporter functions. The use of poly(styrene-co-maleic acid) (SMA) copolymers has allowed their separation in a single step without any exposure to synthetic detergents or artificial lipids. This is a critical development as these agents typically strip away biological lipids, signals, and metabolites from their physiologically-relevant positions on proteins. The resulting SMA lipid particles (SMALPs) represent native nanodiscs that are suitable for elucidation of structures and interactions that occur in vivo. Compatible tools for resolving the contained memteins include X-ray diffraction (XRD), cryo-electron microscopy (cryoEM), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Recent progress shows that memteins are more representative than naked membrane proteins devoid of natural lipid and is driving the development of next generation polymers. [ABSTRACT FROM AUTHOR]

Abstract In this study, we have investigated the lipids surrounding AqpZ, and the effects of a destabilizing mutation W 14 A (Schmidt and Sturgis, 2017) on lipid protein interactions. In a first approach, we used Styrene Maleic Acid copolymer to prepare AqpZ containing nanodiscs, and these were analyzed for their lipid content, investigating both the lipid head-group and acyl-chain compositions. These results were complemented by native mass spectrometry of purified AqpZ in the presence of lipids, to give insights of variations in lipid binding at the surface of AqpZ. In an effort to gain molecular insights, to aid interpretation of these results, we performed a series of coarse grained molecular dynamics simulations of AqpZ, in mixed lipid membranes, and correlated our observations with the experimental measurements. These various results are then integrated to give a clearer picture of the lipid environment of AqpZ, both in the native membrane, and in lipid nanodiscs. We conclude that AqpZ contains a lipid binding-site, at the interface between the monomers of the tetramer, that is specific for cardiolipin. Almost all the cardiolipin, in AqpZ containing nanodiscs, is probably associated with this site. The SMA 3:1 nanodiscs we obtained contain a rather high proportion of lipid, and in the case of nanodiscs containing AqpZ cardiolipin is depleted. This is possibly because, in the membrane, there is little cardiolipin not associated with binding sites on the surface of the different membrane proteins. Surprisingly, we see no evidence for lipid sorting based on acyl chain length, even in the presence of a large hydrophobic mismatch, suggesting that conformational restrictions are energetically less costly than lipid sorting. Graphical Abstract Highlights • Aquaporin Z containing SMALP's are depleted in cardiolipin. • There is no evidence for lipid selection based on acyl-chain structure in AqpZ SMALP's. • Native MS coupled with MD simulations suggest a specific lipid binding site at the tetramer interface. [ABSTRACT FROM AUTHOR]

Neha N Parayath,1 Hayley Nehoff,1 Samuel E Norton,2 Andrew J Highton,2 Sebastien Taurin,1,3 Roslyn A Kemp,2 Khaled Greish1,4 1Department of Pharmacology and Toxicology, 2Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand; 3Department of Obstetrics and Gynecology, University of Utah, Salt Lake City, UT, USA; 4Princess Al-Jawhara Centre for Molecular Medicine, Arabian Gulf University, Manama, Kingdom of Bahrain Abstract: Oral administration of paclitaxel (PTX), a broad spectrum anticancer agent, is challenged by its low uptake due to its poor bioavailability, efflux through P-glycoprotein, and gastrointestinal toxicity. We synthesized PTX nanomicelles using poly(styrene-co-maleic acid) (SMA). Oral administration of SMA-PTX micelles doubled the maximum tolerated dose (60 mg/kg vs 30 mg/kg) compared to the commercially available PTX formulation (PTX [Ebewe]). In a murine orthotopic colon cancer model, oral administration of SMA-PTX micelles at doses 30 mg/kg and 60 mg/kg reduced tumor weight by 54% and 69%, respectively, as compared to the control group, while no significant reduction in tumor weight was observed with 30 mg/kg of PTX (Ebewe). In addition, toxicity of PTX was largely reduced by its encapsulation into SMA. Furthermore, examination of the tumors demonstrated a decrease in the number of blood vessels. Thus, oral delivery of SMA-PTX micelles may provide a safe and effective strategy for the treatment of colon cancer. Keywords: oral delivery, anticancer nanomedicine, CT-26, enhanced permeability and retention (EPR) effect, HUVEC, antiangiogenic

Bee propolis has been used in alternative medicine to treat various diseases. Due to its limited water solubility, it is often used in combination with alcohol solvents, causing skin irritation and immune response. To solve this, the new drug delivery system, based on the lipid nanodiscs of 1,2-dimyristoyl- sn -glycero-3-phosphochline (DMPC) and poly(styrene- alt -maleic acid) (PSMA), were created in an aqueous media. At the excess polymer concentrations, the PSMA/DMPC complexation produced the very fine nanoparticles (18 nm). With the increased molar ratio of styrene to maleic acid (St/MA) in the copolymer structure, the lipid nanodisc showed the improved encapsulation efficiency (EE%), comparing to their corresponding aqueous formulations. The maximum value had reached to around 20% when using the 2:1 PSMA precursor. Based on the cytotoxicity test, these nanoparticles were considered to be non-toxic over the low dose administration region (<78 µg/mL). Instead, they possessed the ability to promote the Vero cell growth. The new PSMA/DMPC nanovesicles could thus be used to improve aqueous solubility and therapeutic effects of poorly water-soluble drugs, thus extending their use in modern therapies.

Highlights • Memtein is coined to describe a membrane protein bound to a layer of biologically relevant, structured lipids. • Devolopment of styrene maleic acid lipid particle (SMALP) technology to prepare native nanodiscs reviewed. • Impact of SMALP and related methods on structural biology and memtein research is explored. Abstract The concept of a memtein as the minimal unit of membrane function is proposed here, and refers to the complex of a membrane protein together with a continuous layer of biological lipid molecules. The elucidation of the atomic resolution structures and specific interactions within memteins remains technically challenging. Nonetheless, we argue that these entities are critical endpoints for the postgenomic era, being essential units of cellular function that mediate signal transduction and trafficking. Their biological mechanisms and molecular compositions can be resolved using native nanodiscs formed by poly(styrene-co-maleic acid) (SMA) copolymers. These amphipathic polymers rapidly and spontaneously fragment membranes into water-soluble discs holding a section of bilayer. This allows structures of complexes found in vivo to be prepared without resorting to synthetic detergents or artificial lipids. The ex situ structures of memteins can be resolved by methods including cryo-electron microscopy (cEM), X-ray crystallography (XRC), NMR spectroscopy and mass spectrometry (MS). Progress in the field demonstrates that memteins are better representations of how biology actually works in membranes than naked proteins devoid of lipid, spurring on further advances in polymer chemistry to resolve their details. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The elucidation of the structures and interactions of proteins and lipids in intact biological membranes remains largely uncharted territory. However, this information is crucial for understanding how organelles are assembled and how transmembrane machines transduce signals. The challenge of seeing how lipids and proteins engage each other in vivo remains difficult but is being aided by a group of amphipathic copolymers that spontaneously fragment native membranes into native nanodiscs. Poly(styrene- co -maleic acid) (SMA) copolymers have proven adept at converting membranes, cells and tissues directly into SMA lipid particles (SMALPs), allowing endogenous lipid: protein complexes to be prepared and analyzed. Unlike other amphipathic polymers such as amphipols, SMALP formation requires no conventional detergents, which typically strip lipid molecules from proteins and can destabilize multimers. A collaborative community of hundreds of investigators known as the SMALP network has emerged to develop and apply new technologies and identify new challenges and design potential solutions. In this contribution, we review recent practices and progress, focusing on novel SMA copolymers, modifications, alternatives and mechanisms. In addition, a brief overview will be provided, with reference to the further contributions to this special issue on the SMALP technology. [ABSTRACT FROM AUTHOR]

Discovering how membrane proteins recognize signals and passage molecules remains challenging. Life depends on compartmentalizing these processes into dynamic lipid bilayers that are technically difficult to work with. Several polymers have proven adept at separating the responsible machines intact for detailed analysis of their structures and interactions. Styrene maleic acid (SMA) co-polymers efficiently solubilize membranes into native nanodiscs and, unlike amphipols and membrane scaffold proteins, require no potentially destabilizing detergents. Here we review progress with the SMA lipid particle (SMALP) system and its impacts including three dimensional structures and biochemical functions of peripheral and transmembrane proteins. Polymers systems are emerging to tackle the remaining challenges for wider use and future applications including in membrane proteomics, structural biology of transient or unstable states, and discovery of ligand and drug-like molecules specific for native lipid-bound states. [ABSTRACT FROM AUTHOR]

The omics disciplines remain largely distinct sciences due to the necessity of separating molecular classes for different assays. For example, water-soluble and lipid bilayer-bound proteins and metabolites are usually studied separately. Nonetheless, it is at the interface between these sciences where biology happens. That is, lipid-interacting proteins typically recognize and transduce signals and regulate the flow of metabolites in the cell. Technologies are emerging to converge the omics. It is now possible to separate intact membrane:protein assemblies (memteins) directly from intact cells or cell membranes. Such complexes mediate complete metabolon, receptor, channel, and transporter functions. The use of poly(styrene-co-maleic acid) (SMA) copolymers has allowed their separation in a single step without any exposure to synthetic detergents or artificial lipids. This is a critical development as these agents typically strip away biological lipids, signals, and metabolites from their physiologically-relevant positions on proteins. The resulting SMA lipid particles (SMALPs) represent native nanodiscs that are suitable for elucidation of structures and interactions that occur in vivo. Compatible tools for resolving the contained memteins include X-ray diffraction (XRD), cryo-electron microscopy (cryoEM), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Recent progress shows that memteins are more representative than naked membrane proteins devoid of natural lipid and is driving the development of next generation polymers.

Engineering natural photosynthesis to address predicted shortfalls in food and energy supply requires a detailed understanding of its molecular basis and the intrinsic photoprotective mechanisms that operate under fluctuating environmental conditions. Long-lived triplet or singlet excited electronic states have the potential to cause photodamage, particularly in the presence of oxygen, and so a variety of mechanisms exist to prevent formation of such states or safely dissipate their energy. Here, we report a dramatic difference in spectral evolution in fully reduced and partially oxidized Rhodobacter sphaeroides reaction centres (RCs) following excitation of the monomeric bacteriochlorophyll (BChl) cofactors at 805 nm. Three types of preparation were studied, including RCs purified as protein/lipid nanodiscs using the copolymer styrene maleic acid. In fully reduced RCs such excitation produces membrane-spanning charge separation. In preparations of partially oxidized RCs the spectroscopic signature of this charge separation is replaced by that of an energy dissipation process, including in the majority sub-population of reduced RCs. This process, which appears to take place on both cofactor branches, involves formation of a BChl+/ bacteriopheophytin- radical pair that dissipates energy via recombination to a vibrationally hot ground state. The possible physiological role of this dissipative process under mildly oxidizing conditions is considered. [ABSTRACT FROM AUTHOR]

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.

The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology. [ABSTRACT FROM AUTHOR]

Cannabinoid receptor agonists are moderately effective at reducing neuropathic pain but are limited by psychoactivity. We developed a styrene maleic acid (SMA) based on the cannabinoid WIN 55,212-2 (WIN) and tested in a rat model of neuropathic pain and in the rotarod test. We hypothesized that miceller preparation can ensure prolonged plasma half-life being above the renal threshold of excretion. Furthermore, SMA-WIN could potentially reduce the central nervous system effects of encapsulated WIN by limiting its transport across the blood-brain barrier. Using the chronic constriction injury model of sciatic neuropathy, the SMA-WIN micelles were efficacious in the treatment of neuropathic pain for a prolonged period compared to control (base WIN). Attenuation of chronic constriction injury-induced mechanical allodynia occurred for up to 8 h at a dose of 11.5 mg/kg of SMA-WIN micelles. To evaluate central effects on motor function, the rotarod assessment was utilized. Results showed initial impairment caused by SMA-WIN micelles to be identical to WIN control for up to 1.5 h. Despite this, the SMA-WIN micelle formulation was able to produce prolonged analgesia over a time when there was decreased impairment in the rotarod test compared with base WIN. [ABSTRACT FROM AUTHOR]

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. [ABSTRACT FROM AUTHOR]

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell "SMALPome", membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be "non-specific" than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Morrison, Heesom, Edler, Doutch, Price, Koumanov and Whitley.)

• Prions are shown to be membrane-dependent rather than prion-only particles. • Native lipid interactions are revealed using native nanodiscs known as SMALPs. • A structured binding site for membrane recognition is discovered using MODA. • The plethora of lipid binding elements are modulated by disease-linked mutations. • Conversion alters and propagation reinforces the membrane interaction surface. Although prions are known as protein-only infectious particles, they exhibit lipid specificities, cofactor dependencies and membrane-dependent activities. Such membrane interactions play key roles in how prions are processed, presented and regulated, and hence have significant functional consequences. The expansive literature related to prion protein interactions with lipids and native nanodiscs is discussed, and provides a unique opportunity to re-evaluate the molecular composition and mechanisms of its infectious and cellular states. A family of crystal and solution structures of prions are analyzed here for the first time using the membrane optimal docking area (MODA) program, revealling the presence of structured binding elements that could mediate specific lipid recognition. A set of motifs centerred around W99, L125, Y169 and Y226 are consistently predicted as being membrane interactive and form an exposed surface which includes α helical, β strand and loop elements involving the prion protein (PrP) structural domain, while the scrapie form is radically different and doubles the size of the membrane interactive site into an extensible surface. These motifs are highly conserved throughout mammalian evolution, suggesting that prions have long been intrinsically attached to membranes at central and N- and C-terminal points, providing several opportunities for stable and specific bilayer interactions as well as multiple complexed orientations. Resistance or susceptibility to prion disease correlates with increased or decreased membrane binding propensity by mutant forms, respectively, indicating a protective role by lipids. The various prion states found in vivo are increasingly resolvable using native nanodiscs formed by styrene maleic acid (SMA) and stilbene maleic acid (STMA) copolymers rather than classical detergents, allowing the endogenous states to be tackled. These copolymers spontaneously fragment intact membranes into water-soluble discs holding a section of native bilayer, and can accommodate prion multimers and mini-fibrils. Such nanodiscs have also proven useful for understanding how β amyloid and α synuclein proteins contribute to Alzheimer's and Parkinson's diseases, providing further biomedical applications. Structural and functional insights of such proteins in styrene maleic acid lipid particles (SMALPs) can be resolved at high resolution by methods including cryo-electron microscopy (cEM), motivating continued progress in polymer design to resolve biological and pathological mechanisms. [ABSTRACT FROM AUTHOR]

Membrane proteins (MPs) are essential to many organisms' major functions. They are notorious for being difficult to isolate and study, and mimicking native conditions for studies in vitro has proved to be a challenge. Lipid nanodiscs are among the most promising platforms for MP reconstitution, but they contain a relatively labile lipid bilayer and their use requires previous protein solubilization in detergent. These limitations have led to the testing of copolymers in new types of nanodisc platforms. Polymer-encased nanodiscs and polymer nanodiscs support functional MPs and address some of the limitations present in other MP reconstitution platforms. In this review, we provide a summary of recent developments in the use of polymers in nanodiscs., (Copyright © 2020 Chen, Majdinasab, Fiori, Liang and Altenberg.)

Engineering natural photosynthesis to address predicted shortfalls in food and energy supply requires a detailed understanding of its molecular basis and the intrinsic photoprotective mechanisms that operate under fluctuating environmental conditions. Long-lived triplet or singlet excited electronic states have the potential to cause photodamage, particularly in the presence of oxygen, and so a variety of mechanisms exist to prevent formation of such states or safely dissipate their energy. Here, we report a dramatic difference in spectral evolution in fully reduced and partially oxidized Rhodobacter sphaeroides reaction centres (RCs) following excitation of the monomeric bacteriochlorophyll (BChl) cofactors at 805 nm. Three types of preparation were studied, including RCs purified as protein/lipid nanodiscs using the copolymer styrene maleic acid. In fully reduced RCs such excitation produces membrane-spanning charge separation. In preparations of partially oxidized RCs the spectroscopic signature of this charge separation is replaced by that of an energy dissipation process, including in the majority sub-population of reduced RCs. This process, which appears to take place on both cofactor branches, involves formation of a BChl + /bacteriopheophytin - radical pair that dissipates energy via recombination to a vibrationally hot ground state. The possible physiological role of this dissipative process under mildly oxidizing conditions is considered.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'., (© 2017 The Author(s).)

Background: Vasectomy is currently the only long-acting contraceptive option available for men, despite increasing demand and potentially significant positive impacts on human health of additional male contraceptive options. Vasalgel ™ is a high molecular weight hydrogel polymer being developed as a non-hormonal long-acting reversible male contraceptive. Vasalgel consists of styrene-alt-maleic acid dissolved in dimethyl sulfoxide, which is distinct from styrene-alt-maleic anhydride materials previously studied., Methods: The goal of the study was to determine the contraceptive efficacy of two test articles with different levels of styrene maleic acid (100 %, and 80 % acid/20 % anhydride). The test articles were injected bilaterally in the vasa deferentia of mature male rabbits. Post-implantation analyses of semen parameters were completed over a 12 month period and compared to baseline measures of sperm concentration, motility and forward progression., Results: Both test articles were effective in blocking the passage of spermatozoa through the vasa deferentia in the 12 subjects completing the study. A significant decrease in sperm concentration occurred following implantation of the test material, with no measurable sperm concentration except for a few samples in one animal that were markedly oligospermic. Vasalgel produced a rapid onset of azoospermia, with no sperm in semen samples collected as early as 29-36 days post-implantation, and was durable over a 12 month period., Conclusion: This study indicated that Vasalgel is an effective non-hormonal long-acting male contraceptive in a rabbit model.