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Cytochrome c oxidase (C c O) 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 C c O 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 C c O. Our data demonstrate that the presence of CO at heme a 3 does not impair the catalytic oxidation of C c O 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 C c O. • In situ gas titrations benchmark reactions of cytochrome c oxidase. • Reactions can be performed using molecular O 2 and without the need for light. • CO ligation does not influence the oxidation of fully-reduced cytochrome c oxidase. • Ligand photolysis or purging in the mixed-valence state are indistinguishable. [ABSTRACT FROM AUTHOR]

Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study.

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic α-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix–helix interactions in lipid-free states or helix–lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop “zip-code”-based delivery for therapeutic and diagnostic applications.

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.

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.

The expansion of native mass spectrometry (MS) methods for both academic and industrial applications has created a substantial need for analysis of large native MS datasets. Existing software tools are poorly suited for high-throughput deconvolution of native electrospray mass spectra from intact proteins and protein complexes. The UniDec Bayesian deconvolution algorithm is uniquely well suited for high-throughput analysis due to its speed and robustness but was previously tailored towards individual spectra. Here, we optimized UniDec for deconvolution, analysis, and visualization of large data sets. This new module, MetaUniDec, centers around a hierarchical data format 5 (HDF5) format for storing datasets that significantly improves speed, portability, and file size. It also includes code optimizations to improve speed and a new graphical user interface for visualization, interaction, and analysis of data. To demonstrate the utility of MetaUniDec, we applied the software to analyze automated collision voltage ramps with a small bacterial heme protein and large lipoprotein nanodiscs. Upon increasing collisional activation, bacterial heme-nitric oxide/oxygen binding (H-NOX) protein shows a discrete loss of bound heme, and nanodiscs show a continuous loss of lipids and charge. By using MetaUniDec to track changes in peak area or mass as a function of collision voltage, we explore the energetic profile of collisional activation in an ultra-high mass range Orbitrap mass spectrometer.ᅟ [ABSTRACT FROM AUTHOR]

Abstract Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms. Graphical abstract Unlabelled Image Highlights • Identification and modulation of molecular ligand selectivity filters in GPCRs. • Agonist binding to GPCRs can be controlled by directed engineering of small domains. • Different recognition mechanisms for the common agonist ET-1 by endothelin receptors • A second disulfide bond stabilizes endothelin B receptor. [ABSTRACT FROM AUTHOR]

Highlights • Integral membrane proteins require a lipid environment for full function. • Membrane proteins may be reconstituted into a variety of lipid systems. • Symmetric reconstitution systems provide access to both sides of a membrane protein. • Liposomal reconstitution systems enable measurement of vectorial substrate movement. • The reconstitution system used will depend on the nature of the functional activity. Abstract Membrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins; namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their substrates. Reconstitution may take many forms and these have been classified into two broad categories. Symmetric systems enable unfettered access to both sides of a bilayer. Compartment containing systems contain a lumen and are ideally suited to measurement of transport processes. The investigator is encouraged to ascertain what aspects of protein function will be undertaken and to apply the most advantageous reconstitution system or systems. It is important to note that the process of reconstitution is not subject to defined protocols and requires empirical optimisation to specific targets. [ABSTRACT FROM AUTHOR]

Abstract: Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b5 (cyt‐b5), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities. [ABSTRACT FROM AUTHOR]

Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs (PLPDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB5). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PLPDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PLPDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB5 measured for pre-loaded PDs and PLPDs prepared from glycolipids extracted from pig and mouse brain revealed that the PLPDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PLPDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS.ᅟ [ABSTRACT FROM AUTHOR]

Abstract: Structural studies on membrane‐anchored proteins containing a transmembrane (TM) helix have been hampered by difficulties in producing these proteins in a natively folded form. Detergents that are required to solubilize the hydrophobic TM helix usually destabilize the soluble domain. Thus, TM helices are removed for structural studies, which neglects the pivotal role of a membrane on protein function. This work presents a versatile strategy for the production of this protein class attached to phospholipid nanodiscs. By inserting the TM‐helix into nanodiscs and a subsequent SortaseA‐mediated ligation of the soluble domain, membrane‐anchored BclxL could be obtained in a folded conformation. This strategy is suitable for high‐resolution structure determination as well as for probing membrane location by NMR. This method will be applicable to a wide range of membrane‐anchored proteins and will be useful to decipher their functional role in a native membrane environment. [ABSTRACT FROM AUTHOR]

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. [ABSTRACT FROM AUTHOR]

Abstract: Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide‐based lipid nanodiscs to “trap” the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein‐induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane. [ABSTRACT FROM AUTHOR]

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Förster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general. [ABSTRACT FROM AUTHOR]

[Display omitted] Curcumin extracted from the rhizomes of Curcuma longa and widely used because of its biological advantages such as anti-inflammatory, anti-cancer, anti-oxidant, anti-ageing, anti-microbial, and wound healing. A wide range of diseases can be diagnosed with the help of curcumin nanoformulations, which have profoundly impacted the pharmaceutical industry. For instance, the anti-cancer effects of curcumin are a highlighted side of curcumin's therapeutic effects. Curcumin, a natural phytochemical agent, can provide an adjuvant when combined with radiotherapy and chemotherapy. Although the unique properties of curcumin make this bioactive substance a versatile therapeutic agent, the lack of appropriate formulation limited the clinical and pharmaceutical application of curcumin. Emerging nanotechnology, particularly nanoparticles, can solve chemical therapy's limitations thanks to its ability to entrap poorly soluble drugs for administration, enhance internalization, and reduce side effects. Among different nanoparticles, nanogels (NGs) with three-dimensional nanonetwork structures and capability covering Both hydrogels and nanoparticles properties could overcome various usual limitations of nanoparticles and enhance the local and systemic drug delivery. Another possible carrier for curcumin is nanodiscs (NDs). NDs which are not spherical, have been paid considerable attention. The concept of the ND constitution is derived from biologically-derived solutions for transferring lipids in the bloodstream. Furthermore, several studies have investigated the efficiency and dependability of curcumin distribution with yeast-based frameworks. Lastly, complex curcumin with transition metals boosts the pharmacological effects of this bioactive molecule, and it is therefore required to raise its bioavailability and solubility in water. In this chapter, we will review the application of NGs, NDs, Yeast cells, and metallo-complexes-based materials in curcumin delivery and briefly evaluate their potential benefits for enhancing curcumin application in the pharmaceutical and clinical setting. [ABSTRACT FROM AUTHOR]

Thermophotovoltaic (TPV) cells convert thermally emitted photons into electrical power using photovoltaic (PV) detectors. To realize highly efficient thermal energy harvesting using TPV conversion, high-temperature stable spectrally-selective emitters are needed. The deployment of TPV technology lags behind conventional solar-PV technology due to the lack of large-scale fabrication of efficient thermal emitters, which would preferentially emit in the PV cell absorption band. In this work, we demonstrate a simple large-area nanofabrication method based on the hole-mask colloidal lithography and sputtering, which allows one to fabricate tungsten (W) nanodisc spectrally-selective emitters (consisting of a metal-insulator-metal configuration) with a high emissivity below the InGaAsSb PV-cell cut-off wavelength of 2.25 μm and a gradually decreasing emissivity (down to < 10%) in the mid-infrared region. Frequency-domain time-domain (FDTD) simulations reveal that the spectral selectivity is achieved due to the localized surface plasmon resonance of W nanodiscs strongly influenced by the insulator thickness. Importantly, the W emitters show thermal stability at temperatures of up to 1100 °C, and emissivity invariance to changes in polarization and incidence angles up to 65°. This work represents a significant step towards the realization of high-temperature stable efficient thermal emitters by a facile and cost-effective fabrication method, thereby promoting the implementation of photonic/plasmonic thermal emitters in the next-generation thermal energy harvesting systems. The method proposed in this study holds potential for scalability; however, empirical evidence to demonstrate this scalability has not yet been established. Subsequent studies are needed to confirm the scalability of the proposed method and its extensive applicability. [Display omitted] • Tungsten nanodisc-based thermal emitters are fabricated using a simple large-area nanofabrication technique. • Tungsten nanodisc emitters exhibit exceptional thermal stability up to elevated temperatures of 1100 °C. • Polarization and angle invariance in the spectral selectivity is observed up to 65° incidence angles. • Tunable spectral selectivity is obtained by changing the spacer layer thickness. [ABSTRACT FROM AUTHOR]

Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability. Here, we show that docosahexaenoic acid (DHA), a key ω−3 polyunsaturated fatty acid in synaptic membranes, enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC. We determined a 3.25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation. The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces. In this previously unobserved conformation, the extracellular-half of the pore-lining M2 is splayed open, reminiscent of the open conformation, while the intracellular-half is constricted, leading to a loss of both water and permeant ions. These findings, in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes, provide novel mechanistic details of desensitization in pentameric channels.

Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches.

A complex cell envelope, composed of a mixture of lipid types including lipopolysaccharides, protects bacteria from the external environment. Clearly, the proteins embedded within the various components of the cell envelope have an intricate relationship with their local environment. Therefore, to obtain meaningful results, molecular simulations need to mimic as far as possible this chemically heterogeneous system. However, setting up such systems for computational studies is far from trivial, and consequently the vast majority of simulations of outer membrane proteins still rely on oversimplified phospholipid membrane models. This work presents an update of CHARMM-GUI Martini Maker for coarse-grained modeling and simulation of complex bacterial membranes with lipopolysaccharides. The qualities of the outer membrane systems generated by Martini Maker are validated by simulating them in bilayer, vesicle, nanodisc, and micelle environments (with and without outer membrane proteins) using the Martini force field. We expect this new feature in Martini Maker to be a useful tool for modeling large, complicated bacterial outer membrane systems in a user-friendly manner. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c -dependent nitric oxide reductase ( c NOR) from Paracoccus ( P. ) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized c NOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O 2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O 2 -binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced c NOR and O 2 , and found that the p K a of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed. [ABSTRACT FROM AUTHOR]

The gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from isobutanol or aqueous solutions of GA-containing nanodiscs (NDs), are investigated using electrospray ionization-ion mobility separation-mass spectrometry (ESI-IMS-MS) and molecular dynamics (MD) simulations. The IMS arrival times measured for (2GA + 2Na) ions from isobutanol reveal three different conformations, with collision cross-sections (Ω) of 683 Å (conformation 1, C1), 708 Å (C2), and 737 Å (C3). The addition of NHCHCO produced (2GA + 2Na) and (2GA + H + Na) ions, with Ω similar to those of C1, C2, and C3, as well as (2GA + 2H), (2GA + 2NH), and (2GA + H + NH) ions, which adopt a single conformation with a Ω similar to that of C2. These results suggest that the nature of the charging agents, imparted by the ESI process, can influence dimer conformation in the gas phase. Notably, the POPC NDs produced exclusively (2GA + 2NH) dimer ions; the DMPC NDs produced both (2GA + 2H) and (2GA + 2NH) dimer ions. While the Ω of (2GA + 2H) is similar to that of C2, the (2GA + 2NH) ions from NDs adopt a more compact structure, with a Ω of 656 Å. It is proposed that this compact structure corresponds to the ion conducting single stranded head-to-head helical GA dimer. These findings highlight the potential of NDs, combined with ESI, for transferring transmembrane peptide complexes directly from lipid bilayers to the gas phase. [ABSTRACT FROM AUTHOR]

Polymer-based nanodiscs are valuable tools in biomedical research that can offer a detergent-free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA-based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer-based nanodiscs are characterized by light scattering, NMR, FT-IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub-micrometer diameter, can be produced by varying the lipid-to-polymer ratio. The small-size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic-alignment of macro-nanodiscs (diameter of > ca. 40 nm) can be exploited for solid-state NMR studies on membrane proteins. [ABSTRACT FROM AUTHOR]

In an attempt to manipulate the biological features of nanomaterials via both anisotropic shape and ligand modification, four types of nanoparticulates with good morphological stability are designed and engineered, including hybrid nanospheres, nanodiscs, and nanodiscs with edge modification or plane modification of octa-arginine (R8) sequence. It is found that the R8 modification anisotropy can trigger huge differences in the endocytosis, intracellular trafficking, and even tissue penetration of nanoparticulates. From plane modification to edge modification of R8, the maximum increase in cell uptake is up to 17-fold, which is much more significant than shape anisotropy alone. On the other hand, six types of different cell lines are investigated to simulate biological microenvironment. It is demonstrated that the maximum difference in cell uptake among six cell lines is 12-fold. Three main driving forces are found to contribute to such bio-nano interactions. Based on the findings of this study, it seems possible to manipulate the biointeraction mode of nanomaterials and its output by regulating their anisotropy in both shape and ligand modification. [ABSTRACT FROM AUTHOR]

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures. [ABSTRACT FROM AUTHOR]

A room temperature reduction-hydrolysis of Fe(III) precursors such as FeCl3 or Fe(acac)3 in various lyotropic liquid crystal phases (lamellar, hexagonal columnar or micellar) formed by a range of ionic or neutral surfactants in H2O is shown to be an effective and mild approach for the preparation of iron oxide (IO) nanomaterials with several morphologies (shapes and dimensions), such as extended thin nanosheets with lateral dimensions of several hundred nanometers as well as smaller nanoflakes and nanodiscs in the tens of nanometers size regime. We will discuss the role of the used surfactants and lyotropic liquid crystal phases as well as the shape and size differences depending upon when and how the resulting nanomaterials were isolated from the reaction mixture. The presented synthetic methodology using lyotropic liquid crystal solvents should be widely applicable to several other transition metal oxides for which the described reduction-hydrolysis reaction sequence is a suitable pathway to obtain nanoscale particles. [ABSTRACT FROM AUTHOR]

Autophosphorylating histidine kinase CheA is central to signaling in bacterial chemotaxis. The kinase donates its phosphoryl group to two response regulators, CheY that controls flagellar rotation and thus motility and CheB, crucial for sensory adaptation. As measured by coupled CheY phosphorylation, incorporation into signaling complexes activates the kinase ∼1000-fold and places it under control of chemoreceptors. By the same assay, receptors modulate kinase activity ∼100-fold as a function of receptor ligand occupancy and adaptational modification. These changes are the essence of chemotactic signaling. Yet, the enzymatic properties affected by incorporation into signaling complexes, by chemoreceptor ligand binding or by receptor adaptational modification are largely undefined. To investigate, we performed steady-state kinetic analysis of autophosphorylation using a liberated kinase phosphoryl-accepting domain, characterizing kinase alone, in isolated core signaling complexes and in small arrays of core complexes assembled in vitro with receptors contained in isolated native membranes. Autophosphorylation in signaling complexes was measured as a function of ligand occupancy and adaptational modification. Activation by incorporation into signaling complexes and modulation in complexes by ligand occupancy and adaptational modification occurred largely via changes in the apparent catalytic rate constant ( kcat). Changes in the autophosphorylation kcat accounted for most of the ∼1000-fold kinase activation in signaling complexes observed for coupled CheY phosphorylation, and the ∼100-fold inhibition by ligand occupancy or modulation by adaptational modification. Our results indicate no more than a minor role in kinase control for simple sequestration of the autophosphorylation substrate. Instead they indicate direct effects on the active site. [ABSTRACT FROM AUTHOR]

The haem-containing mono-oxygenase cytochrome P450 3A4 ( CYP3A4) and its redox partner NADPH-dependent cytochrome P450 oxidoreductase ( CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane-bound in the endoplasmic reticulum ( ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady-state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7-benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono-oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4-nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR- CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR- CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity. [ABSTRACT FROM AUTHOR]

Conformational equilibria of G-protein--coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron--electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ~16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners. [ABSTRACT FROM AUTHOR]

Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis. Taxadiene binding followed type-1 substrate patterns with an observed K D of 2.1 ± 0.4 μM. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) and taxadiene metabolism was measured. Taxadiene metabolism followed Michaelis-Menten kinetics with an observed V max of 30 ± 8 pmol/min/nmol CYP725A4 and a K M of 123 ± 52 μM. Additionally, molecular operating environment (MOE) modeling was performed in order to gain insight into the interactions of taxadiene with CYP725A4 active site. Taken together, we demonstrate the successful expression and purification of the functional membrane-bound plant CYP, CYP725A4, in E. coli. [ABSTRACT FROM AUTHOR]

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD. [ABSTRACT FROM AUTHOR]

Membrane proteins are one of the most challenging and attractive objects in modern structural biology, as they are targets for the majority of medicines. However, studies of membrane proteins are hindered by several obstacles, including their low ability to crystallize, highly dynamic behavior of some of their domains, and need for membrane-like environment. Although solution nuclear magnetic resonance (NMR) is a very powerful technique of structural biology in terms of the amount of provided data, it imposes several limitations on the object under investigation, with the main constraint being related to the size of the object. For this reason, the membrane mimetic has to form particles of small size and simultaneously to properly simulate the bilayer membrane to be applicable for solution NMR spectroscopy. Here we review the recent advances in the field of membrane mimetics for solution NMR studies, discuss the advantages and drawbacks of specific membrane-like environments, and formulate the criteria for the selection of proper environment for a particular membrane protein or domain. [ABSTRACT FROM AUTHOR]

[Display omitted] • Protein synthesis in the cell-free expression system. • Incorporation of the potassium channel ROMK2 in nanodiscs into the lipid bilayers. • Channel activity after reconstitution into free-standing BLMs and tethered BLMs. • Electrochemical measurements of channel activity. 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • EPR of spin-labeled AM2 demonstrates similar conformation in nanodiscs and liposomes. • AM2-liposomes are more permeable to oxygen than AM2-nanodiscs. • Membrane topology of AM2 is similar in both liposomes and nanodiscs. • Cholesterol induces conformational shifts in AM2 in both nanodiscs and liposomes. [ABSTRACT FROM AUTHOR]

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.