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The brain is an exceptionally lipid-rich organ with a very complex lipid composition. Lipids are central in several neuronal processes, including membrane formation and fusion, myelin packing, and lipid-mediated signal transmission. Lipid diversity is associated with the evolution of higher cognitive abilities in primates, is affected by neuronal activity, and is instrumental for synaptic plasticity, illustrating that lipids are not static components of synaptic membranes. Several lines of evidence suggest that the lipid composition of synapses is unique and distinct from other neuronal subcompartments. Here, we delve into the nascent field of synaptoneurolipidomics, offering an overview of current knowledge on the lipid composition of synaptic junctions and technological advances that will allow us to study the impact on synaptic function., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lipid nanodiscs have become a widely used approach for studying membrane proteins thanks to several advantages they offer. They have been especially useful for studying ABC transporters, despite the growing concern about the possible restriction of the conformational changes of the transporters due to the small size of the discs. Here, we performed a systematic study to determine the effect of the nanodisc size on the ATPase activity of model ABC transporters from human, plant, and bacteria. Our data confirm that the activity of the transporters and their response to regulatory molecules is affected by the nanodisc size. Our findings suggest the use of larger membrane scaffold proteins (MSPs), such as MSP2N2 nanodiscs, to minimize alterations caused by the commonly used small MSP1D1., (© 2025 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling., Competing Interests: EB, JM, WW No competing interests declared, (© 2024, Bruguera et al.)

Nanoparticles have the potential to improve disease treatment and diagnosis due to their ability to incorporate drugs, alter pharmacokinetics, and enable tissue targeting. While considerable effort is placed on developing spherical lipid-based nanocarriers, recent evidence suggests that high aspect ratio lipid nanocarriers can exhibit enhanced disease site targeting and altered cellular interactions. However, the assembly of lipid-based nanoparticles into non-spherical morphologies has typically required incorporating additional agents such as synthetic polymers, proteins, lipid-polymer conjugates, or detergents. Here, charged lipid headgroups are used to generate stable discoidal lipid nanoparticles from mixed micelles, which are termed charge-stabilized nanodiscs (CNDs). The ability to generate CNDs in buffers with physiological ionic strength is restricted to lipids with more than one anionic group, whereas monovalent lipids only generate small nanoliposomal assemblies. In mice, the smaller size and anisotropic shape of CNDs promote higher accumulation in subcutaneous tumors than spherical liposomes. Further, the surface chemistry of CNDs can be modified via layer-by-layer (LbL) assembly to improve their tumor-targeting properties over state-of-the-art LbL-liposomes when tested using a metastatic model of ovarian cancer. The application of charge-mediated anisotropy in lipid-based assemblies can aid in the future design of biomaterials and cell-membrane mimetic structures., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Binding of cytochrome c (Cytc) to membranes containing cardiolipin (CL) is of considerable interest because of the importance of this interaction in the early stages of apoptosis. The molecular-level determinants of this interaction are still not well defined and there appear to be species-specific differences in Cytc affinity for CL-containing membranes. Many studies are carried out at low ionic strength far from the 100-150 mM ionic strength within mitochondria. Similarly, most binding studies are done at Cytc concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cytc present in mitochondria. In this study, we evaluate binding of human and yeast Cytc to CL nanodiscs using size exclusion chromatography at 25 μM Cytc concentration and 100 mM ionic strength. We find that yeast Cytc affinity for CL nanodiscs is much stronger than that of human Cytc. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cytc binding to CL nanodiscs than lysines 72 and 73, counter to results at lower ionic strength. Analysis of the electrostatic surface potential of human versus yeast Cytc shows that the positive potential due to lysines 86 and 87 and other nearby lysines (4, 5, 11, 89) is stronger than that due to lysines 72 and 73. In the case of human Cytc the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterisation by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines., Competing Interests: Declaration of competing interest The project was funded by the Dutch Research Council with financial contributions from Batavia Biosciences B.V. and ZoBio B.V., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H + -coupled antiporters EmrE and SugE, the lactose permease LacY, the Na + /H + antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as K M , I MAX , and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Andre Bazzone reports a relationship with Nanion Technologies GmbH that includes employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Due to their fundamental biological importance, membrane proteins (MPs) are attractive targets for drug discovery, with cell surface receptors, transporters, ion channels, and membrane-bound enzymes being of particular interest. However, due to numerous challenges, these proteins present underutilized opportunities for discovering biotherapeutics. Antibodies hold the promise of exquisite specificity and adaptability, making them the ideal candidates for targeting complex membrane proteins. They can target specific conformations of a particular membrane protein and can be engineered into various formats. Generating specific and effective antibodies targeting these proteins is no easy task due to several factors. The antigen's design, antibody-generation strategies, lead optimization technologies, and antibody modalities can be modified to tackle these challenges. The rational employment of cutting-edge lipid nanoparticle systems for retrieving the membrane antigen has been successfully implemented to simplify the mechanism-based therapeutic antibody discovery approach. Despite the highlighted MP production challenges, this review unequivocally underscores the advantages of targeting complex membrane proteins with antibodies and designing membrane protein antigens. Selected examples of lipid nanoparticle success have been illustrated, emphasizing the potential of therapeutic antibody discovery in this regard. With further research and development, we can overcome these challenges and unlock the full potential of therapeutic antibodies directed to target complex MPs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare relevant to this article's content., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nanodiscs (NDs), self-assembled lipid bilayers encircled by membrane scaffold proteins (MSPs), offer a versatile platform for the reconstitution of membrane proteins for structural and biochemical investigations. Saturated, isoprenoid lipids are commonly found in thermophiles and have been associated with thermotolerance. To test whether these lipids confer additional stability on ND-incorporated membrane proteins, this study focuses on the thermal stability of human cytochrome P450 3A4 (CYP3A4) inside NDs composed of different phosphocholine lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). NDs were characterized using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and densitometric SDS-PAGE. CYP3A4-DPhPC-NDs were found to comprise three MSP copies instead of the canonical dimer, as reported before for the empty NDs. Rapid, thermally induced unfolding of CYP3A4 inside NDs measured using circular dichroism and differential scanning fluorimetry (nanoDSF) revealed that the CYP3A4 melting temperature was dependent on ND composition. In POPC and DMPC-CYP3A4-NDs the melting temperature was comparable to CYP3A4 without NDs (59 °C). CYP3A4 in DPhPC-NDs showed an increase in melting temperature of 4 °C. Decline in CYP3A4 integrity as well as ND aggregation and disintegration occur at similar rates for all membrane types when subjected to exposure at 37 °C for several hours. The POPC and DMPC- CYP3A4-NDs show significant lipid loss over time, which is not observed for DPhPC-NDs. The results demonstrate that thermally induced denaturation of protein-NDs is a complex, multifaceted process, which is not represented well by rapid thermal unfolding experiments., Competing Interests: Declaration of competing interest The project was funded by the Dutch Research Council with financial contributions from Batavia Biosciences B.V. and ZoBio B.V., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised. While this detergent-based approach has produced meaningful results in membrane protein research, a lipid environment should be more suitable to recapture the protein's native folding and functions. This protocol describes how to prepare amphipathic membrane scaffold-proteins (MSPs)-based nanodiscs of a cation-coupled melibiose symporter of Salmonella enterica serovar Typhimurium (MelB St ), a member of the major facilitator superfamily. MSPs generate nano-assemblies containing membrane proteins surrounded by a patch of native lipids to better preserve their native conformations and functions. This protocol requires purified membrane protein in detergents, purified MSPs in solution, and detergent-destabilized phospholipids. The mixture of all three components at specific ratios is incubated in the presence of Bio-Beads SM-2 resins, which absorb all detergent molecules, allowing the membrane protein to associate with lipids surrounded by the MSPs. By reconstituting the purified membrane proteins back into their native-like lipid environment, these nanodisc-like particles can be directly used in cryo-EM single-particle analysis for structure determination and other biophysical analyses. It is noted that nanodiscs may potentially limit the dynamics of membrane proteins due to suboptimal nanodisc size compared to the native lipid bilayer. Key features • This protocol was built based on the method originally developed by Sligar et al. [1] and modified for a specific major facilitator superfamily transporter • This protocol is robust and reproducible • Lipid nanodiscs can increase membrane protein stability, and reconstituted transporters in lipid nanodiscs can regain function if their function is compromised using detergents • The reconstituted lipids nanodisc can be used for cryo-EM single-particle analysis., Competing Interests: Competing interestsThe authors declare no competing financial interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

Chemosensory membrane proteins such as G-protein-coupled receptors (GPCRs) drive flavor perception of food formulations. To achieve this, a detailed understanding of the structure and function of these membrane proteins is needed, which is often limited by the extraction and purification methods involved. The proposed nanodisc methodology helps overcome some of these existing challenges such as protein stability and solubilization along with their reconstitution from a native cell-membrane environment. Being well-established in structural biology procedures, nanodiscs offer this elegant solution by using, e.g., a membrane scaffold protein (MSP) or styrene-maleic acid (SMA) polymer, which interacts directly with the cell membrane during protein reconstitution. Such derived proteins retain their biophysical properties without compromising the membrane architecture. Here, we seek to show that these lipidic systems can be explored for insights with a focus on chemosensory membrane protein morphology and structure, conformational dynamics of protein-ligand interactions, and binding kinetics to answer pending questions in flavor research. Additionally, the compatibility of nanodiscs across varied (labeled or label-free) techniques offers significant leverage, which has been highlighted here.

Tumor heterogeneity remains a significant obstacle in cancer therapy due to diverse cells with varying treatment responses. Cancer stem-like cells (CSCs) contribute significantly to intratumor heterogeneity, characterized by high tumorigenicity and chemoresistance. CSCs reside in the depth of the tumor, possessing low reactive oxygen species (ROS) levels and robust antioxidant defense systems to maintain self-renewal and stemness. A nanotherapeutic strategy is developed using tumor-penetrating peptide iRGD-modified high-density lipoprotein (HDL)-mimetic nanodiscs (IPCND) that ingeniously loaded with pyropheophorbide-a (Ppa), bis (2-hydroxyethyl) disulfide (S-S), and camptothecin (CPT) by synthesizing two amphiphilic drug-conjugated sphingomyelin derivatives. Photoactivatable Ppa can generate massive ROS which as intracellular signaling molecules effectively shut down self-renewal and trigger differentiation of the CSCs, while S-S is utilized to deplete GSH and sustainably imbalance redox homeostasis by reducing ROS clearance. Simultaneously, the depletion of GSH is accompanied by the release of CPT, which leads to subsequent cell death. This dual strategy successfully disturbed the redox equilibrium of CSCs, prompting their differentiation and boosting the ability of CPT to kill CSCs upon laser irradiation. Additionally, it demonstrated a synergistic anti-cancer effect by concurrently eliminating therapeutically resistant CSCs and bulk tumor cells, effectively suppressing tumor growth in CSC-enriched heterogeneous colon tumor mouse models., (© 2024 Wiley‐VCH GmbH.)

Nanodisc technology is increasingly being used in structural, biochemical and biophysical studies of membrane proteins. The computational approaches have revealed many important features of nanodisc assembly, structures and dynamics. Therefore, we reviewed the application of computational approaches, especially molecular modeling and molecular dyncamics (MD) simulations, to characterize nanodiscs, including the structural models, assembly and disassembly, protocols for modeling, structural properties and dynamics, and protein-lipid interactions in nanodiscs. More amazing computational studies about nanodiscs are looked forward to in the future., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures., (Copyright © 2024. Published by Elsevier Inc.)

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The decreasing efficacy of antiviral drugs due to viral mutations highlights the challenge of developing a single agent targeting multiple strains. Using host cell viral receptors as competitive inhibitors is promising, but their low potency and membrane-bound nature have limited this strategy. In this study, the authors show that angiotensin-converting enzyme 2 (ACE2) in a planar membrane patch can effectively neutralize all tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that emerged during the COVID-19 pandemic. The ACE2-incorporated membrane patch implemented using nanodiscs replicated the spike-mediated membrane fusion process outside the host cell, resulting in virus lysis, extracellular RNA release, and potent antiviral activity. While neutralizing antibodies became ineffective as the SARS-CoV-2 evolved to better penetrate host cells the ACE2-incorporated nanodiscs became more potent, highlighting the advantages of using receptor-incorporated nanodiscs for antiviral purposes. ACE2-incorporated immunodisc, an Fc fusion nanodisc developed in this study, completely protected humanized mice infected with SARS-CoV-2 after prolonged retention in the airways. This study demonstrates that the incorporation of viral receptors into immunodisc transforms the entry gate into a potent virucide for all current and future variants, a concept that can be extended to different viruses., (© 2024 Wiley‐VCH GmbH.)

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo ., Importance: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies., Competing Interests: The authors declare no conflict of interest.

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.

Discoidal lipid nanoparticles (LNPs) called Nanodiscs (NDs) are derived from human high-density lipoprotein (HDL). Such biomimetics are ideally suited for the stabilization and delivery of pharmaceuticals, including chemicals, bio-active proteins and vaccines. The stability and circulation lifetimes of reconstituted HDL nanoparticles, including NDs, are variable. Lipids found in thermophilic archaea and bacteria are prime candidates for the stabilization of LNPs. We report the thermal stability of NDs prepared with lipids that differ in saturation, have either ether- or ester linkages between the fatty acid and glycerol backbone or contain isoprenoid fatty acid tails (phytanyl lipids). NDs with two saturated fatty acids show a much greater long-term thermostability than NDs with an unsaturated fatty acid. Ether fatty acid linkages, commonly found in thermophiles, did not improve stability of NDs compared to ester fatty acid linkages when using saturated lipids. NDs containing phytanyl and saturated alkyl fatty acids show similar stability at 37 °C. NDs assembled with phytanyl lipids contain three copies of the membrane scaffolding protein as opposed to the canonical dimer found in conventional NDs. The findings present a strong basis for the production of thermostable NDs through the selection of appropriate lipids and are likely broadly applicable to LNP development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marcellus Ubbink reports financial support was provided by The Dutch Research Council (NWO). Marcellus Ubbink reports a relationship with Batavia Biosciences BV that includes: funding grants. Marcellus Ubbink reports a relationship with ZoBio BV that includes: funding grants. The authors disclose a potential competing interest due to an ongoing patent filing related to the topics discussed in this study. The project was funded by the Dutch Research Council with financial contributions by Batavia Biosciences B.V. and ZoBio B.V., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo , suggesting a potential sepsis treatment.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Biomimetic delivery systems (BDSs), inspired by the intricate designs of biological systems, have emerged as a groundbreaking paradigm in nanomedicine, offering unparalleled advantages in therapeutic delivery. These systems, encompassing platforms such as liposomes, protein-based nanoparticles, extracellular vesicles, and polysaccharides, are lauded for their targeted delivery, minimized side effects, and enhanced therapeutic outcomes. However, the translation of BDSs from research settings to clinical applications is fraught with challenges, including reproducibility concerns, physiological stability, and rigorous efficacy and safety evaluations. Furthermore, the innovative nature of BDSs demands the reevaluation and evolution of existing regulatory and ethical frameworks. This review provides an overview of BDSs and delves into the multifaceted translational challenges and present emerging solutions, underscored by real-world case studies. Emphasizing the potential of BDSs to redefine healthcare, we advocate for sustained interdisciplinary collaboration and research. As our understanding of biological systems deepens, the future of BDSs in clinical translation appears promising, with a focus on personalized medicine and refined patient-specific delivery systems.

Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O 2 to H 2 O at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited R 2 CO state as well as the fully-reduced R 4 and R 4 CO states and induced the "active ready" oxidized state O H . These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O 2 , the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a 3 does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the R 2 CO state, the release of the CO ligand upon purging with inert gas yield a product that is indistinguishable from photolysis-induced states. The observed changes at heme a 3 in the catalytic binuclear center (BNC) result from the loss of CO and are unrelated to electronic excitation upon illumination. Based on our experiments, we re-evaluate the assignment of marker bands that appear in time-resolved photolysis and perfusion-induced experiments on CcO., Competing Interests: Declaration of competing interest 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., (Copyright © 2023. Published by Elsevier B.V.)

Many membrane proteins form functional complexes that are either homo- or hetero-oligomeric. However, it is challenging to characterize membrane protein oligomerization in intact lipid bilayers, especially for polydisperse mixtures. Native mass spectrometry of membrane proteins and peptides inserted in lipid nanodiscs provides a unique method to study the oligomeric state distribution and lipid preferences of oligomeric assemblies. To interpret these complex spectra, we developed novel data analysis methods using macromolecular mass defect analysis. Here, we provide an overview of how mass defect analysis can be used to study oligomerization in nanodiscs, discuss potential limitations in interpretation, and explore strategies to resolve these ambiguities. Finally, we review recent work applying this technique to studying formation of antimicrobial peptide, amyloid protein, and viroporin complexes with lipid membranes., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.

Environmental nutrients control bacterial biofilm homeostasis, by regulating the intracellular levels of c-di-GMP. One component transducers can sense different classes of small molecules through a periplasmic domain; the nutrient recognition triggers the subsequent regulation of the downstream cytosolic diguanylate cyclase (GGDEF) or phosphodiesterase (EAL) domains, via transmembrane helix(ces), to finally change c-di-GMP levels.Protein studies on such transducers have been mainly carried out on isolated domains due to the presence of the transmembrane portion. Nevertheless, the cleavage of GGDEF and EAL-containing proteins could be detrimental since both tertiary and quaternary structures could be allosterically controlled; to by-pass this limitation, studies on the corresponding full-length proteins are highly desired.We have in silico selected a GGDEF-EAL transducer from Dyella thiooxydans (ann. A0A160N0B7), whose periplasmic binding domain was predicted to bind to arginine, a nutrient often associated with chronic infections and biofilm. This protein has been used as an in vitro tool for the identification of the best approach for its isolation, including (i) protein engineering to produce a water-soluble version via QTY (Glutamine, Threonine, and Tyrosine) code or (ii) nanodiscs assembly. The results on this "prototype" may represent the proof-of-concept for future isolation of other transmembrane proteins sharing the same architecture, including more complex nutrient-based transducers controlling c-di-GMP levels., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland A.)

Tumor infarction therapy is a promising antitumor strategy with the advantages of taking a short therapy duration, less risk of resistance, and effectiveness against a wide range of tumor types. However, its clinical application is largely hindered by tumor recurrence in the surviving rim and the potential risk of thromboembolic events due to nonspecific vasculature targeting. Herein, a neovasculature-targeting synthetic high-density lipoprotein (sHDL) nanodisc loaded with pyropheophorbide-a and camptothecin (CPN) was fabricated for photoactivatable tumor infarction and synergistic chemotherapy. By manipulating the anisotropy in ligand modification of sHDL nanodiscs, CPN modified with neovaculature-targeting peptide on the planes (PCPN) shows up to 7-fold higher cellular uptake compared with that around the edge (ECPN). PCPN can efficiently bind to endothelial cells of tumor vessels, and upon laser irradiation, massive local thrombus can be induced by the photodynamic reaction to deprive nutrition supply. Meanwhile, CPT could be released in response to the tumor reductive environment, thus killing residual tumor cells in the surviving rim to inhibit recurrence. These findings not only offer a powerful approach of synergistic cancer therapy but also suggest the potential of plane-modified sHDL nanodiscs as a versatile drug delivery nanocarrier.

Membrane scaffold protein nanodiscs (MSPNDs) are an invaluable tool for improving purified membrane protein (MP) stability and activity compared to traditional micellar methods, thus enabling an increase in high-resolution MP structures, particularly in concert with cryogenic electron microscopy (cryo-EM) approaches. In this review we highlight recent advances and breakthroughs in MSPND methodology and applications. We also introduce and discuss saposin-lipoprotein nanoparticles (salipros) and copolymer nanodiscs which have recently emerged as authentic MSPND alternatives. We compare the advantages and disadvantages of MSPNDs, salipros, and copolymer nanodisc technologies to highlight potential opportunities for using each platform for MP purification and characterization., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain. MS2-L mediates bacterial cell lysis through the formation of large lesions in the cell envelope, but further details of this mechanism as a prerequisite for applied bioengineering studies are lacking. The chaperone DnaJ is proposed to modulate MS2-L activity, whereas other cellular targets of MS2-L are unknown. Methods: Here, we provide a combined in vitro and in vivo overexpression approach to reveal molecular insights into MS2-L action and its interaction with DnaJ. Full-length MS2-L and truncated derivatives were synthesized cell-free and co-translationally inserted into nanodiscs or solubilized in detergent micelles. By native liquid bead ion desorption mass spectrometry, we demonstrate that MS2-L assembles into high oligomeric states after membrane insertion. Results: Oligomerization is directed by the transmembrane domain and is impaired in detergent environments. Studies with truncated MS2-L derivatives provide evidence that the soluble domain acts as a modulator of oligomer formation. DnaJ strongly interacts with MS2-L in membranes as well as in detergent environments. However, this interaction affects neither the MS2-L membrane insertion efficiency nor its oligomerization in nanodisc membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in E. coli cells. Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed by disruption of the peptidoglycan layer and disintegration of the inner membrane. Conclusion: MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric interface of both peptides is located within their transmembrane domains. We propose a potential function of the higher-order assembly of small phage toxins in membrane disintegration and cell lysis., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2023.)

Membrane proteins are a highly relevant class of biological molecules and comprise ∼60% of current drug targets. Before being analyzed by structural, biochemical, and biophysical methods, membrane proteins must first be extracted from cellular membranes - often using detergents. Detergent-extracted membrane proteins are amenable to analysis by structural, biochemical, and biophysical techniques. In certain cases, however, detergents can disturb native protein conformations and/or biological activity. This has led to the development of membrane mimetics, which stabilize membrane proteins in a native membrane-like environment that is water-soluble and detergent-free. This review provides an overview of recent developments in the membrane mimetic field, with a focus on nanodiscs, Saposin lipid nanoparticles (SapNPs), peptidiscs, and SMA lipid particles (SMALPs) - and highlights their utility for supporting biophysical, biochemical, and structural characterization of membrane proteins and complexes., (© 2023 The Author(s).)

Oleanolic acid derivative DKS26 has hypolipidemic, islet, and hepatoprotective effects. However, high lipophilicity and low water solubility led to DKS26 extremely low oral bioavailability. Herein, lipid-based nanocarriers, including lipid nanodiscs (sND/DKS26) and liposomes (sLip/DKS26), are prepared to improve DKS26 oral absorption. In comparison to free DKS26 (5.81%), the absolute oral bioavailabilities are significantly increased to 29.47% (sND/DKS26) and 37.25% (sLip/DKS26) without detectable toxicity or immunogenicity even after repeated administrations. Both sND/DKS26 and sLip/DKS26 significantly reduce the feeding glucose level and the AUC of OGTT in db/db diabetic mice. Aiding by the newly developed scFv-based nanocarrier separation methods, no intact nanocarriers are detected in blood circulation after oral administration, suggesting that both formulations are unable to penetrate the intestinal epithelium. They enhance DKS26 absorption mainly by improving intestinal cell uptake and rapid intracellular release of the payload. Since pre-existing anti-PEG is widely detected in humans, the present oral absorption pathway of both nanocarriers successfully avoids unfavorable immunological responses after interaction with anti-PEG antibodies. The application of lipid-based nanocarriers paves an efficient and safe avenue for the clinical translation and application of poorly soluble therapeutics derived from traditional Chinese medicine., (© 2023 Wiley-VCH GmbH.)

The influenza A M2 protein (AM2) is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle including viral assembly and budding. An atomic-level conformational understanding of this key player in the influenza life cycle could inform new antiviral strategies. For conformational studies of complex systems like the AM2 membrane protein, a multipronged approach using different biophysical methods and different model membranes is a powerful way to incorporate complementary data and achieve a fuller, more robust understanding of the system. However, one must be aware of how the sample composition required for a particular method impacts the data collected and how conclusions are drawn. In that spirit, we systematically compared the properties of AM2 in two different model membranes: nanodiscs and liposomes. Electron paramagnetic spectroscopy of spin-labeled AM2 showed that the conformation and dynamics were strikingly similar in both AM2-nanodiscs and AM2-liposomes consistent with similar conformations in both model membranes. Analysis of spin labeled lipids embedded in both model membranes revealed that the bilayer in AM2-liposomes was more fluid and permeable to oxygen than AM2-nanodiscs with the same lipid composition. Once the difference in the partitioning of the paramagnetic oxygen relaxation agent was taken into account, the membrane topology of AM2 appeared to be the same in both liposomes and nanodiscs. Finally, functionally relevant AM2 conformational shifts previously seen in liposomes due to the addition of cholesterol were also observed in nanodiscs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kathleen P. Howard reports financial support was provided by National Institutes of Health., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The angiotensin-converting enzyme 2 (ACE2), as a functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for assessing potential hosts and treatments. However, many studies are based on its truncated version but not full-length structure. Indeed, a single transmembrane (TM) helix presents in the full-length ACE2, influencing its interaction with SARS-CoV-2. Therefore, synthesis of the full-length ACE2 is an urgent requirement. Here, cell-free membrane protein synthesis systems (CFMPSs) are constructed for full-length membrane proteins. MscL is screened as a model among ten membrane proteins based on their expression and solubility. Next, CFMPSs are constructed and optimized based on natural vesicles, vesicles with four membrane proteins removed or two chaperonins added, and 37 types of nanodiscs. They all increase membrane protein solubility to over 50%. Finally, the full-length ACE2 of 21 species are successfully expressed with yields between 0.4 and 0.9 mg mL -1 . The definite functional differences from the truncated version suggest that the TM region affects ACE2's structure and function. CFMPSs can be extended to more membrane proteins, paving the way for further applications., (© 2023 Wiley-VCH GmbH.)

The renal-outer-medullary‑potassium (ROMK2) channel modulates potassium transport in the kidney. It has been postulated that the ROMK2 is the pore-forming subunit of the mitochondrial ATP-sensitive potassium channel as a mediator of cardioprotection. In this study, cell-free synthesis of the ROMK2 was performed in presence of membrane scaffold protein (MSP1D1) nanodiscs. Activity measurements were achieved after channel reconstitution into the planar lipid bilayer and tethered bilayer lipid membranes. Both methods allowed for monitoring of channel function, verified with channel blocking and activation/re-activation experiments. The primary function of the mitochondrial potassium channels is to regulate the potential of the mitochondrial membrane, which allows them to play an important role in cytoprotection. This work focuses on obtaining the ROMK2 using a cell-free expression system, followed by the incorporation of the channel protein into the lipid bilayer and studying the influence of voltage changes and molecular modulators on channel activity. Channel activity was measured after its reconstitution into two models of lipid bilayers - BLM (Bilayer Lipid Membrane) and tBLM (Tethered Bilayer Lipid Membrane) deposited on a solid gold electrode. These two model membranes and electrochemical measurements made it possible to measure the flux of K + ions in the presence of channel modulators., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli . Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.

Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles.

KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self-association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self-assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self-association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5-α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge-reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane., (© 2023 Wiley-VCH GmbH.)

Integral membrane proteins (IMPs) perform a range of diverse functions and their dysfunction underlies numerous pathological conditions. Consequently, IMPs constitute most drug targets, and the elucidation of their mechanism of action has become an intense field of research. Historically, IMP studies have relied on their extraction from membranes using detergents, which have the potential to perturbate their structure and dynamics. To circumnavigate this issue, an array of membrane mimetics has been developed that aim to reconstitute IMPs into native-like lipid environments that more accurately represent the biological membrane. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a versatile tool for probing protein dynamics in solution. The continued development of HDX-MS methodology has allowed practitioners to investigate IMPs using increasingly native-like membrane mimetics, and even pushing the study of IMPs into the in vivo cellular environment. Consequently, HDX-MS has come of age and is playing an ever-increasingly important role in the IMP structural biologist toolkit. In the present mini-review, we discuss the evolution of membrane mimetics in the HDX-MS context, focusing on seminal publications and recent innovations that have led to this point. We also discuss state-of-the-art methodological and instrumental advancements that are likely to play a significant role in the generation of high-quality HDX-MS data of IMPs in the future., (© 2023 The Author(s).)

P-glycoprotein (Pgp) is a multidrug transporter that uses the energy from ATP binding and hydrolysis to export from cells a wide variety of hydrophobic compounds including anticancer drugs, and mediates the bioavailability and pharmacokinetics of many drugs. Lipids and cholesterol have been shown to modulate the substrate-stimulated ATPase activity of purified Pgp in detergent solution and the substrate transport activity after reconstitution into proteoliposomes. While lipid extracts from E. coli , liver or brain tissues generally support well Pgp's functionality, their ill-defined composition and high UV absorbance make them less suitable for optical biophysical assays. On the other hand, studies with defined synthetic lipids, usually the bilayer-forming phosphatidylcholine with or without cholesterol, are often plagued by low ATPase activity and low binding affinity of Pgp for drugs. Drawing from the lipid composition of mammalian plasma membranes, we here investigate how different head groups modulate the verapamil-stimulated ATPase activity of purified Pgp in detergent-lipid micelles and compare them with components of E. coli lipids. Our general approach was to assay modulation of verapamil-stimulation of ATPase activity by artificial lipid mixtures starting with the bilayer-forming palmitoyloyl-phosphatidylcholine (POPC) and -phosphatidylethanolamine (POPE). We show that POPC/POPE supplemented with sphingomyelin (SM), cardiolipin, or phosphatidic acid enhanced the verapamil-stimulated activity (V max ) and decreased the concentration required for half-maximal activity (EC 50 ). Cholesterol (Chol) and more so its soluble hemisuccinate derivative cholesteryl hemisuccinate substantially decreased EC 50 , perhaps by supporting the functional integrity of the drug binding sites. High concentrations of CHS (>15%) resulted in a significantly increased basal activity which could be due to binding of CHS to the drug binding site as transport substrate or as activator, maybe acting cooperatively with verapamil. Lastly, Pgp reconstituted into liposomes or nanodiscs displayed higher basal activity and sustained high levels of verapamil stimulated activity. The findings establish a stable source of artificial lipid mixtures containing either SM and cholesterol or CHS that restore Pgp functionality with activities and affinities similar to those in the natural plasma membrane environment and will pave the way for future functional and biophysical studies., 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 © 2023 Tran, Bui, Jaramillo-Martinez, Weber, Zhang and Urbatsch.)

Lipid-bilayer nanodiscs and liposomes have been developed to stabilize membrane proteins in order to study their structures and functions. Nanodiscs are detergent-free, water-soluble, and size-controlled planar phospholipid-bilayer platforms. On the other hand, liposomes are curved phospholipid-bilayer spheres with an aqueous core used as drug delivery systems and model membrane platforms for studying cellular activities. A long-standing challenge is the generation of a homogenous and monodispersed lipid-bilayer system with a very wide range of dimensions and curvatures (elongation, bending, and twisting). A DNA-origami template provides a way to control the shapes, sizes, and arrangements of lipid bilayers via enforcing the assembly of lipid bilayers within the cavities created by DNA nanostructures. Here, we provide a concise overview and discuss how to design planar and curved lipid-bilayer membranes by using DNA-origami nanostructures as templates. Finally, we will discuss the potential applications of DNA-origami nanostructures in the structural and functional studies of large membrane proteins and their complexes., Competing Interests: MN is listed as an inventor on a patent number PCT/US2017/042307 for the covalently circularized nanodiscs. The remaining author declares 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 © 2023 Elbahnasawy and Nasr.)

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., (© 2023. The Author(s).)

Bax is a major player in the mitochondrial pathway of apoptosis, by making the Outer Mitochondrial Membrane (OMM) permeable to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not yield Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag. Purified wild-type Bax (BaxWT) and a constitutively active mutant (BaxP168A) displayed biochemical properties that were in line with previous characterizations following their expression in yeast and human cells followed by their reconstitution into liposomes. Both Bax variants were then reconstituted in nanodiscs. Size exclusion chromatography, dynamic light scattering and transmission electron microscopy showed that nanodiscs formed with BaxP168A were larger than nanodiscs formed with BaxWT. This was consistent with the hypothesis that BaxP168A was reconstituted in nanodiscs as an active oligomer., Competing Interests: Declaration of competing interest 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., (Copyright © 2022. Published by Elsevier B.V.)

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Residual dipolar couplings (RDCs) are increasingly used for high-throughput NMR-based structural studies and to provide long-range angular constraints to validate and refine structures of various molecules determined by X-ray crystallography and NMR spectroscopy. RDCs of a given molecule can be measured in an anisotropic environment that aligns in an external magnetic field. Here, we demonstrate the first application of polymer-based nanodiscs for the measurement of RDCs from nucleic acids. Polymer-based nanodiscs prepared using negatively charged SMA-EA polymer and zwitterionic DMPC lipids were characterized by size-exclusion chromatography, 1 H NMR, dynamic light-scattering, and 2 H NMR. The magnetically aligned polymer-nanodiscs were used as an alignment medium to measure RDCs from a 13 C/ 15 N-labeled fluoride riboswitch aptamer using 2D ARTSY-HSQC NMR experiments. The results showed that the alignment of nanodiscs is stable for nucleic acids and nanodisc-induced RDCs fit well with the previously determined solution structure of the riboswitch. These results demonstrate that SMA-EA-based lipid-nanodiscs can be used as a stable alignment medium for high-resolution structural and dynamical studies of nucleic acids, and they can also be applicable to study various other biomolecules and small molecules in general.

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Cell-free expression enables direct cotranslational insertion of G protein coupled receptors (GPCRs) and other membrane proteins into the defined membrane environments of nanodiscs. This technique avoids GPCR contacts with detergents and allows rapid identification of lipid effects on GPCR function as well as fast screening of receptor derivatives. Critical steps of conventional GPCR preparation from cellular membranes followed by detergent-based reconstitution into nanodisc membranes are thus eliminated. We report the efficient cotranslational insertion of full-length human β 1 -adrenergic receptor and of a truncated derivative into preformed nanodisc membranes. Their biochemical characterization revealed significant differences in lipid requirements, dimer formation and ligand binding activity. The truncated receptor showed a higher affinity to most tested ligands, in particular in presence of choline-containing lipids. However, introducing the naturally occurring G389R polymorphism in the full-length receptor resulted into an increased affinity to the antagonists alprenolol and carvedilol. Receptor quality was generally improved by coexpression with the agonist isoproterenol and the percentage of the ligand binding active fraction was twofold increased. Specific coupling of full-length and truncated human receptors in nanodisc membranes to Mini-Gα s protein as well as to purified G s heterotrimer could be demonstrated and homogeneity of purified GPCR/G s protein complexes in nanodiscs was demonstrated by negative stain single particle analysis., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Structure determination of membrane proteins has been a long-standing challenge to understand the molecular basis of life processes. Detergents are widely used to study the structure and function of membrane proteins by various experimental methods, and the application of membrane mimetics is also a prevalent trend in the field of cryo-EM analysis. This review focuses on the widely-used detergents and corresponding properties and structures, and also discusses the growing interests in membrane mimetic systems used in cryo-EM studies, providing insights into the role of detergent alternatives in structure determination.

Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes., 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 © 2022 Umbach, Levin, Neumann, Steinmetzer, Dötsch and Bernhard.)