Search

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Explores different approaches for expressing, purifying, and labeling GPCRs both in vitro and in live cell environments. • Discusses specific sample preparation methods for single-molecule fluorescence microscopy. • Examines the unique challenges involved in each stage of sample preparation for single-molecule GPCR experiments. • Highlights recent applications of single-molecule TIRF microscopy in investigating different classes of human GPCRs. 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. [ABSTRACT FROM AUTHOR]

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 puri fied nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negativestain 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). AlphaFoldpredicted 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bitterness elicits unpleasant sensations in humans, which can hinder the acceptance of foods and medication adherence. Therefore, identifying and masking bitter tastes is crucial for developing palatable foods and promoting medication compliance in the food and pharmaceutical industries. To achieve this, employing agonism and antagonism of bitter taste receptors as effective strategies at the molecular level is essential. In this study, a bioelectronic tongue is developed to characterize the agonism and antagonism of bitter taste receptors. The human bitter taste receptors hTAS2R16 and hTAS2R31 are produced using an Escherichia coli expression system and reconstituted into nanodiscs (NDs). Subsequently, hTAS2R16‐ and hTAS2R31‐NDs are immobilized on the surface of graphene field‐effect transistors (FETs) to construct bioelectronic tongues. The developed system sensitively detected the bitter agonists, salicin and saccharin, at concentrations as low as 100 fM, with high selectivity in real‐time. The dose‐dependent curves shifted and K values decreased by the antagonists of hTAS2R16 and hTAS2R31, indicating antagonism‐based masking of bitter taste. Therefore, the developed bioelectronic tongue holds promise for identifying bitter tastes and evaluating the masking of bitterness based on the agonism and antagonism of hTAS2Rs. [ABSTRACT FROM AUTHOR]

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-dimyristoylsn-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Combining native mass spectrometry and nanodiscs reveals the oligomeric states of membrane proteins and peptides inside lipid bilayers. • Mass defect analysis can be used to better understand the stoichiometry and lipid preferences of membrane proteins and peptides. • This review describes how to perform macromolecular mass defect analysis, limitations of the technique, strategies for interpretation, and applications to studying the membrane biophysics of antimicrobial peptides, viroporins, and amyloid proteins. 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. [ABSTRACT FROM AUTHOR]

Nanodiscs have become an extremely valuable tool for membrane protein (MP) research. The first nanodiscs, membrane scaffold protein nanodiscs (MSPNDs), have seen substantial technological development over the past decade, and salipros and copolymer nanodiscs have recently emerged as promising alternatives to MSPNDs. Recent advances include the introduction of circular MSPNDs, improvements to robust nanodisc method development, and increased breakthrough applications for MP structures and biophysical binding studies. Salipros are able to adapt to the size of different MPs by using a single scaffold variant. Although very recently established, salipro methodology and applications have seen rapid development. Copolymer nanodiscs can incorporate MPs in their endogenous lipids via extracting the MP directly from the cell membranes. Three groups of copolymers – styrene–maleic acid (SMA), diisobutylene maleic acid (DIBMA), and polymethacrylate polymer (PMA) – have been employed with varying success to stabilize MPs in an active state. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this work, ZnO has been synthesized with a variety of nanomorphologies, including nanorods (NRs), nanodiscs (NDs), and nanorods/nanodiscs (NRs/NDs), to enhance CO2 gas detection at room temperature. The ZnO nanostructures were made by combining the successive ionic layer adsorption and reaction (SILAR) strategy and the chemical bath deposition (CBD) method. The time of CBD varied from 6 to 12 h. Several techniques, including X-ray diffraction (XRD) spectroscopy, energy-dispersive X-ray (EDAX) spectrometry, optical spectrophotometer, and field emission scanning electron microscopy (FE-SEM), were used to investigate the manufactured ZnO nanostructures. The FE-SEM demonstrates that by increasing the deposition period of CBD from 6 to 12 h, the shape of ZnO nanostructures changed from NRs/NDs to NDs. According to the XRD, all ZnO nanostructured samples exhibit hexagonal wurtzite structures with (002) preferred orientation. Additionally, the crystallite size along orientation (002) increases from 63 to 65 nm as the deposition duration increases from 6 to 12 h. The bandgap of ZnO was reduced from 3.62 to 3.31 eV. When the deposition time is increased from 6 to 12 h, the sensitivity increases from 8.46 to 28.7%, the detection limit rises from 4.65 to 9.95 SCCM, and the limit of quantification rises from 15.52 to 33.16 SCCM. Moreover, the ZnO @ 12 h sensors has excellent selectivity as well since it reacts to CO2 with a higher response sensitivity than it does to other gases like hydrogen and ammonia. [ABSTRACT FROM AUTHOR]

Binding of cytochrome c (Cyt c) 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 Cyt c 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 Cyt c concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cyt c present in mitochondria. In this study, we evaluate binding of human and yeast Cyt c to CL nanodiscs using size exclusion chromatography at 25 μM Cyt c concentration and 100 mM ionic strength. We find that yeast Cyt c affinity for CL nanodiscs is much stronger than that of human Cyt c. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cyt c 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 Cyt c 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 Cyt c the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A. Yeast iso-1-cytochrome c binds more strongly to cardiolipin nanodiscs than human cytochrome c at physiological ionic strength because the charge distribution near site A (lysines 72, 73, 86, 87) is more uniformly positive. Mutagenesis studies on site A lysines suggest that N- and C-terminal helix lysines contribute to electrostatic binding. [Display omitted] • Human cytochrome c does not bind to cardiolipin nanodiscs at 100 mM ionic strength. • Yeast iso-1-cytochrome c binds to cardiolipin nanodiscs at 100 mM ionic strength. • The electrostatic surface potential is stronger for yeast versus human cytochrome c. • Site A lysines 86 and 87 are most important for binding at 100 mM ionic strength. [ABSTRACT FROM AUTHOR]

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, 1H NMR, dynamic light-scattering, and 2H NMR. The magnetically aligned polymer-nanodiscs were used as an alignment medium to measure RDCs from a 13C/15N-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. [ABSTRACT FROM AUTHOR]

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. • Demonstrated methodology from viral glycoprotein cloning to Nanodisc incorporation. • Utilized static light scattering for detailed characterisation of multi-component Nanodiscs. • Reconstituted full-length Influenza A hemagglutinin (HA) in Nanodiscs as single trimers. [ABSTRACT FROM AUTHOR]

[Display omitted] • Transporter proteins are essential for cellular function, but their functions are poorly characterized due to the lack of direct assays. • New workflow for transporter functional characterization combines cell-free transporter protein expression and solid supported membrane-based electrophysiology. • The workflow can be executed in five days. • Five transporters from SMR, MFS, Nha, and MC families were functionally expressed and analyzed. • The assay can provide: substrate specificity, kinetic parameters, pH dependency, and mechanistic insights. 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Phytanyl chain Nanodiscs contain three scaffold proteins, offering accessible lipid area for membrane protein reconstitution. • Cytochrome P450 3A4 has a higher melting temperature in phytanyl Nanodiscs than in Nanodiscs containing linear acyl lipids. • Nanodisc thermal stability was measured over time using orthogonal techniques to assess constituent fates and stability. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Nanodisc-based platforms emerged as a more advanced approach to antibody discovery and drug development. • Advantages and limitations of current protein expression systems for membrane proteins are described. • Successful implementations of nanodisc-based platforms for antibody discovery against membrane proteins are reviewed. • Prospects of nanodisc-based antibody and vaccine discovery strategies are outlined. [ABSTRACT FROM AUTHOR]

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation. [ABSTRACT FROM AUTHOR]

Comparisons of G protein-coupled receptor (GPCR) complexes with agonists and antagonists based on X-ray crystallography and cryo-electron microscopy structure determinations show differences in the width of the orthosteric ligand binding groove over the range from 0.3 to 2.9 Å. Here, we show that there are transient structure fluctuations with amplitudes up to at least 6 Å. The experiments were performed with the neurokinin 1 receptor (NK1R), a GPCR of class A that is involved in inflammation, pain, and cancer. We used 19F-NMR observation of aprepitant, which is an approved drug that targets NK1R for the treatment of chemotherapy-induced nausea and vomiting. Aprepitant includes a bis-trifluoromethyl-phenyl ring attached with a single bond to the core of the molecule; 19F-NMR revealed 180° flipping motions of this ring about this bond. In the picture emerging from the 19F-NMR data, the GPCR transmembrane helices undergo large-scale floating motions in the lipid bilayer. The functional implication is of extensive promiscuity of initial ligand binding, primarily determined by size and shape of the ligand, with subsequent selection by unique interactions between atom groups of the ligand and the GPCR within the binding groove. This second step ensures the wide range of different efficacies documented for GPCR-targeting drugs. The NK1R data also provide a rationale for the observation that diffracting GPCR crystals are obtained for complexes with only very few of the ligands from libraries of approved drugs and lead compounds that bind to the receptors. [ABSTRACT FROM AUTHOR]

Many antibody‐based antivirals, including broadly neutralizing antibodies (bnAbs) against various influenza virus strains, suffer from limited potency. A booster of the antiviral activity of an antibody is expected to facilitate development of antiviral therapeutics. In this study, a nanodisc (ND), a discoidal lipid bilayer encircled by membrane scaffold proteins, is engineered to provide virucidal properties to antibodies, thereby augmenting their antiviral activity. NDs carrying the Fc‐binding peptide sequence form an antibody‐ND complex (ANC), which can co‐endocytose into cells infected with influenza virus. ANC efficiently inhibits endosome escape of viral RNA by dual complimentary mode of action. While the antibody moiety in an ANC inhibits hemagglutinin‐mediated membrane fusion, its ND moiety destroys the viral envelope using free hemagglutinins that are not captured by antibodies. Providing virus‐infected host cells with the ability to self‐eliminate by the synergistic effect of ANC components dramatically amplifies the antiviral efficacy of a bnAb against influenza virus. When the efficacy of ANC is assessed in mouse models, administration of ANCs dramatically reduces morbidity and mortality compared to bnAb alone. This study is the first to demonstrate the novel nanoparticle ANC and its role in combating viral infections, suggesting that ANC is a versatile platform applicable to various viruses. [ABSTRACT FROM AUTHOR]

Using the density functional theory, we calculate electronic states of various nanoribbons and nanodiscs formed from selected two-dimensional materials, such as graphene, silicene, and hexagonal boron nitride. The main objective of the analysis is a search for zero-energy states in such systems, which is an important issue as their presence indicates certain topological properties associated with chirality. The analysis is also supported by calculating transport properties. [ABSTRACT FROM AUTHOR]

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins. [ABSTRACT FROM AUTHOR]

Lipids play major roles in basic cellular functions. Their analysis, therefore, gained importance; however, it is complicated by the high complexity of natural lipidomes. To overcome this challenge, pre‐separation of the lipids by thin‐layer chromatography (TLC) and high‐resolution mass spectrometry (MS) are often employed. Here, we explore Coomassie staining for TLC‐separated phospholipids and provide an extraction protocol of stained lipids for subsequent MS analysis. We exemplify our approach by analyzing lipid mixtures varying in complexity and found that TLC pre‐separation increases the number of identified lipid species and lipid classes. In addition, we identify and quantify lipids from polymer nanodiscs. In summary, Coomassie staining of TLC‐separated lipids is well‐suited for phospholipids, is compatible with MS, does not require specialized equipment and can be performed independently of subsequent MS experiments. We envision many future applications of our workflow. [ABSTRACT FROM AUTHOR]

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins. [ABSTRACT FROM AUTHOR]

A novel nanodisc‐based immobilization method was developed for high‐efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged‐nanodiscs were prepared in the form of self‐assembly of lipid‐protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid‐to‐membrane scaffold protein molar ratio of 60:1, uniformly regular‐shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of –19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged‐nanodiscs were successfully immobilized onto Ni‐chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc‐immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc‐immobilized chromatography method is promising for the one‐step purification and reconstitution of membrane protein. [ABSTRACT FROM AUTHOR]

Membrane proteins (MPs) play a pivotal role in cellular function and are therefore predominant pharmaceutical targets. Although detailed understanding of MP structure and mechanistic activity is invaluable for rational drug design, challenges are associated with the purification and study of MPs. This review delves into the historical developments that became the prelude to currently available membrane mimetic technologies before shining a spotlight on polymer nanodiscs. These are soluble nanosized particles capable of encompassing MPs embedded in a phospholipid ring. The expanding range of reported amphipathic polymer nanodisc materials is presented and discussed in terms of their tolerance to different solution conditions and their nanodisc properties. Finally, the analytical scope of polymer nanodiscs is considered in both the demonstration of basic nanodisc parameters as well as in the elucidation of structures, lipid–protein interactions, and the functional mechanisms of reconstituted membrane proteins. The final emphasis is given to the unique benefits and applications demonstrated for native nanodiscs accessed through a detergent free process. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Provided a new method for preparing covalent annular nanodiscs. • The new covalent annular nanodiscs exhibit good homogeneity. • The new covalent annular nanodiscs have better stability than traditional nanodiscs. • Assembled membrane proteins using new covalent annular nanodiscs. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Nanodiscs: a crucial biomembrane mimetic system. • Computational insights into nanodiscs. • Review of nanodisc-related computational efforts. • Shedding light on advancements and diverse perspectives of computational methods. [ABSTRACT FROM AUTHOR]

Although lipid nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non‐ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo‐oligosaccharides/inulin is reported. We show the stability of lipid nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic‐alignment properties. The reported results also demonstrate that the non‐ionic polymers extract membrane proteins with unprecedented efficiency. [ABSTRACT FROM AUTHOR]

Although lipid nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non‐ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo‐oligosaccharides/inulin is reported. We show the stability of lipid nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic‐alignment properties. The reported results also demonstrate that the non‐ionic polymers extract membrane proteins with unprecedented efficiency. [ABSTRACT FROM AUTHOR]

This study focuses on analyzing the noncovalent interaction patterns between three lipid‐nanodisc‐forming polymers and nitroxide radicals which are used as small organic tracer molecules. Besides the negatively charged polymers diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) (2:1), the solvation behavior of a newly synthesized zwitterionic styrene/maleic amide sulfobetaine copolymer named SMA‐sulfobetaine (SB) is characterized. The applied spin probes vary in their respective chemical structure, allowing the report of different local micropolarities and nanoscopic regions by recording temperature‐dependent continuous‐wave electron paramagnetic resonance (CW EPR) spectra. In combination with light scattering experiments, a nanoscopic interpretation of the dominant polymer/guest molecule interaction patterns is provided. The results indicate that in SMA and DIBMA, ionic interactions dominate the interaction patterns with other molecules. In SMA‐SB, the zwitterionic side chains mainly induce a dynamic assembly with guest molecules due to weaker noncovalent interactions. Depending on the applied spin probe, temperature‐dependent CW EPR measurements reveal nanoscopic cloud points depending on the interaction patterns with SMA‐SB which can occur more than 20 °C below its macroscopically observed upper critical solution temperature. Finally, the detailed dissection of interaction patterns may provide a better understanding that may even allow tuning the polymers' properties for use in lipid nanodisc formation. [ABSTRACT FROM AUTHOR]

The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane‐bound toxin ΦX174‐E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174‐E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell‐free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174‐E synthesis, resulting in the efficient production of a functional full‐length toxin and engineered derivatives. Pre‐assembled nanodiscs were used to study ΦX174‐E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174‐E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174‐E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174‐E assembles into high‐order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174‐E transmembrane domain with the peptidyl‐prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174‐E soluble domain for the generation of a membrane insertion competent toxin. [ABSTRACT FROM AUTHOR]

1,4‐Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L‐type voltage‐gated Ca2+ (Cav) channels. DHP compounds exhibit chirality‐specific antagonistic or agonistic effects. The structure of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo‐EM structures of nanodisc‐embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)‐(+)‐Bay K8644, and a titration of its agonistic enantiomer (S)‐(−)‐Bay K8644 at resolutions of 2.9–3.4 Å. The amlodipine‐bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)‐(−)‐Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration. [ABSTRACT FROM AUTHOR]

1,4‐Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L‐type voltage‐gated Ca2+ (Cav) channels. DHP compounds exhibit chirality‐specific antagonistic or agonistic effects. The structure of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo‐EM structures of nanodisc‐embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)‐(+)‐Bay K8644, and a titration of its agonistic enantiomer (S)‐(−)‐Bay K8644 at resolutions of 2.9–3.4 Å. The amlodipine‐bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)‐(−)‐Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration. [ABSTRACT FROM AUTHOR]

New software, called Marbles, is introduced that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the contribution of the nanodisc to the SAXS intensity through a semi‐analytical model, while the embedded membrane protein is treated as a set of beads, similarly to as in well known ab initio methods. The reliability and flexibility of this approach is proved by benchmarking the code, implemented in C++ with a Python interface, on a toy model and two proteins with very different geometry and size. [ABSTRACT FROM AUTHOR]

Solar-driven highly efficient photocatalytic decomposition of toxic organic contaminants using magnetically separable α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO ternary hybrid nanodiscs is reported. α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO ternary hybrid nanostructures were synthesized by microwave-assisted co-precipitation and simple co-precipitation methods and well characterized by XRD, micro-Raman, FESEM and UV–vis spectroscopy. FESEM micrographs revealed nanodiscs in case of microwave-assisted co-precipitation whereas nanoparticles and their aggregates were formed under co-precipitation combined with calcination. XRD and Raman studies confirmed the hybrid nature of prepared α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO nanostructures. Photocatalytic performance of α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO hybrid nanostructures was investigated by carrying out the photodegradation of organic dyes MB and MG under solar light illumination. The prepared α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO ternary hybrid magnetic nanodiscs decomposed MB and MG dyes in only 32 and 24 min, respectively. α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO hybrid nanodiscs showed excellent photocatalytic performance together with reusability and easy magnetic separation demonstrating its suitability for solar-driven photocatalytic water purification applications. In-situ scavenger studies showed •OH radicals are the main active radicals in solar-driven photocatalysis by α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO nanodiscs. The tentative mechanism of growth of α-Fe 2 O 3 /ZnFe 2 O 4 /ZnO ternary hybrid nanodiscs and the photocatalytic mechanism are discussed. [ABSTRACT FROM AUTHOR]

Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed. [ABSTRACT FROM AUTHOR]

The prototypic multidrug (Mdr) transporter MdfA from Escherichia coli efflux chemically‐ dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (O o) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (I f) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distances measured by double electron-electron resonance (DEER) between pairs of spin labels in MdfA, reconstituted in nanodiscs, with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (O c /I c) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large-scale conformational changes. Rather, we identified a substrate-responsive lateral gate, which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access. Unlabelled Image • We investigated the conformational states of the multidrug transporter MdfA, embedded in a lipid bilayer. • We found that the resting state of MdfA is a flexible outward-closed/inward-closed conformation. • We identified substrate-induced movements between TMs that are uncoupled from the conceptual rigid-body movements in MFS transporters. • We describe a putative lateral gate that allows the recruitment of lipophilic drugs from the inner leaflet of the membrane. • We conclude that MdfA does not follow the canonical rocker-switch models of MFS transporters. [ABSTRACT FROM AUTHOR]

Cellular membranes have long served as an inspiration for nanomaterial research. The preparation of ultrathin polydopamine (PDA) films with integrated protein pores containing phospholipids and an embedded domain of a membrane protein glycophorin A as simplified cell membrane mimics is reported. Large area, ultrathin PDA films are obtained by electropolymerization on gold surfaces with 10–18 nm thickness and dimensions of up to 2.5 cm2. The films are transferred from gold to various other substrates such as nylon mesh, silicon, or substrates containing holes in the micrometer range, and they remain intact even after transfer. The novel transfer technique gives access to freestanding PDA films that remain stable even at the air interfaces with elastic moduli of ≈6–12 GPa, which are higher than any other PDA films reported before. As the PDA film thickness is within the range of cellular membranes, monodisperse protein nanopores, so‐called "nanodiscs," are integrated as functional entities. These nanodisc‐containing PDA films can serve as semi‐permeable films, in which the embedded pores control material transport. In the future, these simplified cell membrane mimics may offer structural investigations of the embedded membrane proteins to receive an improved understanding of protein‐mediated transport processes in cellular membranes. [ABSTRACT FROM AUTHOR]

Summary: Recently, two‐dimensional (2D) metal chalcogenides are gaining considerable attention for its potential employment in energy storage, catalysis, electronic, and sensing devices. Vanadium disulfide (VS2) from the 2D family gathers intensive attention for its application as promising electrocatalysts in hydrogen evolution reaction (HER). Here, we present a simple, cost‐effective hydrothermal technique for the synthesis of nanodiscs (NDs)‐like VS2 structures. They were characterized by X‐ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) and evaluated as electrochemical catalysts in hydrogen evolution reaction (HER). NDs‐like structures made up of thin nanoplates with active sites were found to play a huge role on their electrochemical properties. The prepared VS2 NDs show highly efficient electro catalytic performances with a Tafel slope of 79 mV dec−1. In addition, the prepared VS2 NDs exhibited high stability in acidic solution without any sign for degradation even after 500 continuous potential cycles. The excellent HER performance and long‐term durability suggest the VS2 NDs to serve as an promising and inexpensive HER electro catalyst. [ABSTRACT FROM AUTHOR]

Precision metabolomics and quantification for cost-effective rapid diagnosis of disease are the key goals in personalized medicine and point-of-care testing. At present, patients are subjected to multiple test procedures requiring large laboratory equipment. Microelectronics has already made modern computing and communications possible by integration of complex functions within a single chip. As More than Moore technology increases in importance, integrated circuits for densely patterned sensor chips have grown in significance. Here, we present a versatile single complementary metal–oxide–semiconductor chip forming a platform to address personalized needs through on-chip multimodal optical and electrochemical detection that will reduce the number of tests that patients must take. The chip integrates interleaved sensing subsystems for quadruple-mode colorimetric, chemiluminescent, surface plasmon resonance, and hydrogen ion measurements. These subsystems include a photodiode array and a single photon avalanche diode array with some elements functionalized to introduce a surface plasmon resonance mode. The chip also includes an array of ion sensitive field-effect transistors. The sensor arrays are distributed uniformly over an active area on the chip surface in a scalable and modular design. Bio-functionalization of the physical sensors yields a highly selective simultaneous multiple-assay platform in a disposable format. We demonstrate its versatile capabilities through quantified bio-assays performed on-chip for glucose, cholesterol, urea, and urate, each within their naturally occurring physiological range. [ABSTRACT FROM AUTHOR]

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viralmembrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion. [ABSTRACT FROM AUTHOR]

Membrane protein research suffers from the drawback that detergents, which are commonly used to solubilize integral membrane proteins (IMPs), often lead to protein instability and reduced activity. Recently, lipid nanodiscs (NDs) and saposin-lipoprotein particles (Salipro) have emerged as alternative carrier systems that keep membrane proteins in a native-like lipidic solution environment and are suitable for biophysical and structural studies. Here, we systematically compare nanodiscs and Salipros with respect to long-term stability as well as activity and stability of the incorporated membrane protein using the ABC transporter MsbA as model system. Our results show that both systems are suitable for activity measurements as well as structural studies in solution. Based on our results we suggest screening of different lipids with respect to activity and stability of the incorporated IMP before performing structural studies. [ABSTRACT FROM AUTHOR]

• Biochemistry of plant cell wall biosynthesis lacks high-throughput methods. • Overview of new high-throughput technologies to benefit plant cell wall biosynthesis. • Use of protein array technology in enzyme assays and protein–protein interaction. • Adapting recent advances in analytical chemistry for rapid product identification. Progress in the functional biochemical analysis of plant glycosyltransferases (GTs) has been slow because plant GTs are generally membrane proteins, operate as part of larger, multimeric complexes, and utilize a vast complexity of substrate acceptors. Therefore, the field would benefit from development of adequate high throughput expression as well as product detection and characterization techniques. Here we review current approaches to tackle such obstacles and suggest a new path forward: nucleic acid programmable protein arrays (NAPPA) with liquid sample desorption ionization (LS-DESI-MS) mass spectrometry. NAPPA utilizes in vitro transcription and translation to produce epitope-tagged fusion proteins from cloned GT cDNAs. LS-DESI is a soft ionization technique that allows rapid and sensitive MS-based product characterization in situ. Coupling both approaches provides the opportunity to examine individual GT functions as well as protein–protein interactions. Furthermore, advances in automated oligosaccharide synthesis and lipid nanodisc technology should allow testing of plant GT activity in presence of numerous substrate acceptors and lipid environments in a high throughput fashion. Thus, NAPPA-DESI-MS has great potential to make headway in biochemical characterization of the large number of plant GTs. [ABSTRACT FROM AUTHOR]