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Passive antibody therapies, typically administered via parenteral routes, have played a crucial role in the initial response to the COVID-19 pandemic. However, the ongoing evolution of SARS-CoV-2 has revealed significant limitations of this approach, primarily due to mutational escape and the inadequate delivery of antibodies to the upper respiratory tract. To overcome these challenges, we propose a novel prophylactic strategy involving the intranasal delivery of an antibody in combination with an Fc-binding nanodisc. This nanodisc, engineered to specifically bind to the Fc regions of IgG antibodies, served two key functions: extending the antibody's half-life in the larynx and trachea, and enhancing its neutralization efficacy. Notably, Sotrovimab, an FDA-approved monoclonal antibody that has experienced a significant decline in neutralizing potency due to viral evolution, exhibited robust antiviral activity when complexed with the nanodisc against all tested Omicron variants. Furthermore, the Fc-binding nanodisc significantly boosted the antiviral efficacy of the soluble angiotensin-converting enzyme 2 (sACE2) Fc fusion protein, which possesses broad but modest antiviral activity. In ACE2 transgenic mice, the Fc-binding nanodisc protected better than sACE2-Fc alone with two more log reduction in lung viral titer. Therefore, the intranasal Fc-binding nanodisc offers a promising and powerful approach to counteract the diminished antiviral activity of neutralizing antibodies caused by mutational escape, effectively restoring antiviral efficacy against various evolving SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Background: Vaccine development against Chlamydia, a prevalent sexually transmitted infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccines based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP). Methods: Cell-free protein synthesis (CFPS) technology is an attractive approach to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provide a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade, we have been working toward the production and characterization of MOMP-NLP complexes for vaccine testing. Results: The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scattering (DLS), which confirm the formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1. Conclusion: Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential to elicit robust immune responses. However, further research is warranted to optimize vaccine formulations further, validate efficacy against Chlamydia trachomatis, and better understand the underlying mechanisms of immune response. [ABSTRACT FROM AUTHOR]

RAS proteins are peripheral membrane GTPases that activate multiple downstream effectors for cell proliferation and differentiation. The formation of a signaling RAS–RAF complex at the plasma membrane is implicated in a quarter of all human cancers; however, the underlying mechanism remains unclear. In this work, nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses to determine the structure of a hetero‐tetrameric complex comprising KRAS and the RAS‐binding domain (RBD) and cysteine‐rich domain (CRD) of activated RAF1 are employed. The binding of the RBD or RBD–CRD differentially alters the dimerization modes of KRAS on both anionic and neutral membranes, validated by interface‐specific mutagenesis. Notably, the RBD binding allosterically generated two distinct KRAS dimer interfaces in equilibrium, favored by KRAS free and in complex with the RBD–CRD, respectively. Additional interactions of the CRD with both KRAS protomers are mutually cooperative to stabilize a new dimer configuration of KRAS bound to the RBD–CRD. The RAF binding sequentially alters KRAS dimerization, providing new insights into RAF activation, including a configurational transition of the KRAS dimer to provide an interaction site for the CRD and release the autoinhibited RAF complex. These methods are applicable to many other signaling protein complexes on the membrane. [ABSTRACT FROM AUTHOR]

Transient homo-dimerization of the RAS GTPase at the plasma membrane has been shown to promote the mitogen-activated protein kinase (MAPK) signaling pathway essential for cell proliferation and oncogenesis. To date, numerous crystallographic studies have focused on the well-defined GTPase domains of RAS isoforms, which lack the disordered C-terminal membrane anchor, thus providing limited structural insight into membrane-bound RAS molecules. Recently, lipid-bilayer nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses have revealed several distinct structures of the membrane-anchored homodimers of KRAS, an isoform that is most frequently mutated in human cancers. The KRAS dimerization interface is highly plastic and altered by biologically relevant conditions, including oncogenic mutations, the nucleotide states of the protein, and the lipid composition. Notably, PRE-derived structures of KRAS homodimers on the membrane substantially differ in terms of the relative orientation of the protomers at an "α–α" dimer interface comprising two α4–α5 regions. This interface plasticity along with the altered orientations of KRAS on the membrane impact the accessibility of KRAS to downstream effectors and regulatory proteins. Further, nanodisc platforms used to drive KRAS dimerization can be used to screen potential anticancer drugs that target membrane-bound RAS dimers and probe their structural mechanism of action. [ABSTRACT FROM AUTHOR]

Numerous potential drug targets, including G-protein-coupled receptors and ion channel proteins, reside on the cell surface as multi-pass membrane proteins. Unfortunately, despite advances in engineering technologies, engineering biologics against multi-pass membrane proteins remains a formidable task. In this review, we focus on the different methods used to prepare/present multi-pass transmembrane proteins for engineering target-specific biologics such as antibodies, nanobodies and synthetic scaffold proteins. The engineered biologics exhibit high specificity and affinity, and have broad applications as therapeutics, probes for cell staining and chaperones for promoting protein crystallization. We primarily cover publications on this topic from the past 10 years, with a focus on the different formats of multi-pass transmembrane proteins. Finally, the remaining challenges facing this field and new technologies developed to overcome a number of obstacles are discussed. [ABSTRACT FROM AUTHOR]

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin–phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness. [ABSTRACT FROM AUTHOR]

Integral membrane proteins are important components of a cell. Their structural and functional studies require production of milligram amounts of proteins, which nowadays is not a routine process. Cell-free protein synthesis is a prospective approach to resolve this task. However, there are few known membrane mimetics that can be used to synthesize active membrane proteins in high amounts. Here, we present the application of commercially available "Facade" detergents for the production of active rhodopsin. We show that the yield of active protein in lipid bicelles containing Facade-EM, Facade-TEM, and Facade-EPC is several times higher than in the case of conventional bicelles with CHAPS and DHPC and is comparable to the yield in the presence of lipid-protein nanodiscs. Moreover, the effects of the lipid-to-detergent ratio, concentration of detergent in the feeding mixture, and lipid composition of the bicelles on the total, soluble, and active protein yields are discussed. We show that Facade-based bicelles represent a prospective membrane mimetic, available for the production of membrane proteins in a cell-free system. [ABSTRACT FROM AUTHOR]

The development of novel and active photocatalysts to industrialize photocatalysis technology is still a challenging task. In this work, a novel method is presented to prepare TiO2/SiO2 NSs by covering SiO2 nanospheres (NSs) with titanate-nanodiscs (TNDs) followed by calcination. In this regard, SiO2 NSs are first synthesized and then TNDs are deposited on the SiO2 NSs using a layer-by-layer deposition technique. The morphology of the prepared samples is analyzed via SEM and TEM analyses before and after the deposition. The analysis of metal (Cu, Pt, and Ni) loading on calcined TNDs/SiO2 NSs reveals the highest specific surface area (109 m2/g), absorption wavelength extension (up to 420 nm), and photocatalytic activity for the Cu-loaded sample. In addition, studying the effect of metal content shows that loading 3% Cu leads to the highest photocatalytic activity. Finally, it is demonstrated that H2S treatment can improve the photocatalytic activity by around 15%. These findings suggest the calcined TNDs/SiO2 NSs are a versatile photocatalyst with potential applications in other processes such as hydrogen production and CO2 valorization. [ABSTRACT FROM AUTHOR]

High-density lipoproteins (HDL) are complex endogenous nanoparticles involved in important functions such as reverse cholesterol transport and immunomodulatory activities, ensuring metabolic homeostasis and vascular health. The ability of HDL to interact with a plethora of immune cells and structural cells places it in the center of numerous disease pathophysiologies. However, inflammatory dysregulation can lead to pathogenic remodeling and post-translational modification of HDL, rendering HDL dysfunctional or even pro-inflammatory. Monocytes and macrophages play a critical role in mediating vascular inflammation, such as in coronary artery disease (CAD). The fact that HDL nanoparticles have potent anti-inflammatory effects on mononuclear phagocytes has opened new avenues for the development of nanotherapeutics to restore vascular integrity. HDL infusion therapies are being developed to improve the physiological functions of HDL and to quantitatively restore or increase the native HDL pool. The components and design of HDL-based nanoparticles have evolved significantly since their initial introduction with highly anticipated results in an ongoing phase III clinical trial in subjects with acute coronary syndrome. The understanding of mechanisms involved in HDL-based synthetic nanotherapeutics is critical to their design, therapeutic potential and effectiveness. In this review, we provide a current update on HDL-ApoA-I mimetic nanotherapeutics, highlighting the scope of treating vascular diseases by targeting monocytes and macrophages. [ABSTRACT FROM AUTHOR]

Curcumin, a natural polyphenol derived from the spice turmeric (Curcuma longa), contains antioxidant, anti-inflammatory, and anti-cancer properties. However, curcumin bioavailability is inherently low due to poor water solubility and rapid metabolism. Here, we further refined for use curcumin incorporated into "biomimetic" nanolipoprotein particles (cNLPs) consisting of a phospholipid bilayer surrounded by apolipoprotein A1 and amphipathic polymer scaffolding moieties. Our cNLP formulation improves the water solubility of curcumin over 30-fold and produces nanoparticles with ~350 µg/mL total loading capacity for downstream in vitro and in vivo applications. We found that cNLPs were well tolerated in AG05965/MRC-5 human primary lung fibroblasts compared to cultures treated with curcumin solubilized in DMSO (curDMSO). Pre-treatment with cNLPs of quiescent G0/G1-phase MRC-5 cultures improved cell survival following 137Cs gamma ray irradiations, although this finding was reversed in asynchronously cycling log-phase cell cultures. These findings may be useful for establishing cNLPs as a method to improve curcumin bioavailability for administration as a radioprotective and/or radiomitigative agent against ionizing radiation (IR) exposures in non-cycling cells or as a radiosensitizing agent for actively dividing cell populations, such as tumors. [ABSTRACT FROM AUTHOR]

The cytoplasmic domain of receptor tyrosine kinases (RTKs) plays roles as a kinase and a protein scaffold; however, the allocation of these two functions is not fully understood. Here, we analyzed the assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied members of RTKs, by combining single-pair fluorescence resonance energy transfer (FRET) imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (T654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias toward oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of T654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which T654 phosphorylation causes a switch of the major function of EGFR from kinase-activating dimers to scaffolding oligomers. [ABSTRACT FROM AUTHOR]

Background: Combination antiretroviral therapy has significantly advanced HIV-1 infection treatment. However, HIV-1 remains persistent in the brain; the inaccessibility of the blood–brain barrier allows for persistent HIV-1 infections and neuroinflammation. Nanotechnology-based drug carriers such as nanodiscoidal bicelles can provide a solution to combat this challenge. Methods: This study investigated the safety and extended release of a combination antiretroviral therapy drug (tenofovir)-loaded nanodiscs for HIV-1 treatment in the brain both in vitro and in vivo. Result: The nanodiscs entrapped the drug in their interior hydrophobic core and released the payload at the desired location and in a controlled release pattern. The study also included a comparative pharmacokinetic analysis of nanodisc formulations in in vitro and in vivo models. Conclusion: The study provides potential applications of nanodiscs for HIV-1 therapy development. [ABSTRACT FROM AUTHOR]

ZnSe nanodisks:Ti3C2 MXene complex was prepared for the first time. Based on its remarkable photoelectrochemical performance, combined with the enzyme-free toehold-mediated strand displacement reaction, a photoelectrochemical biosensor for the detection of the non-small-cell cancer biomarker ctDNA KRAS G12D was developed. ZnSe nanodisks were in situ grown on Ti3C2 MXene surface by two-step hydrothermal method. The high conductivity and adjustable band gap of MXene significantly enhanced the photoelectric response of ZnSe. Subsequently, the photoelectrochemical biosensor was prepared by combining with the signal amplification function of p-aminophenol and the enzyme-free toehold-mediated strand displacement reaction on the modified ITO electrode surface. Under the optimized conditions, the linear detection range is 0.5 ~ 100.0 fM, and the detection limit is 0.2 fM, which realizes the sensitive detection of KRAS G12D. The photoelectrochemical biosensor constructed opens up a new pathway for the preparation of new Mxene-based composite materials and the research of photoelectrochemical biosensor. Nucleic acid liquid biopsy with ZnSe nanodisks:Ti3C2 MXene photoelectroactive modified electrode [ABSTRACT FROM AUTHOR]

The plasmonic material properties of aluminum allow active plasmon resonances extending from the blue color in the visible range to the ultraviolet (UV) region of the spectrum. Whereas Al is usually avoided for applications of plasmonics due to its losses in the infrared spectrum region. In this work, the study of the scatter and absorption of disk nanoantennas (DNAs) using various types of materials Au, Ag, and Al is accomplished by using the CST microwave studio suite simulation. The results showed that Al can offer good plasmonic properties when DNA radius is 25 nm to 125 nm at 20 nm height and working wavelengths longer than 800 nm in the near-infrared (NIR) region. Al produces negative plasmonic features around 800 nm wavelength due to the interband transition in the imaginary part of epsilon. For Au and Ag, the plasmonic characteristics rapidly decayed when the DNA radius was higher than 60 nm, but in contrast, Al offers good plasmonic features at these large dimensions of DNAs. This extended response of Al in UV, visible, and NIR, incorporated with its low cost, natural abundance, low native oxide, and amenability to industrial processes, could make Al an extremely promising plasmonic metal candidate for energy applications. [ABSTRACT FROM AUTHOR]

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self‐assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native‐like bilayer environment that maintain a target's functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function. [ABSTRACT FROM AUTHOR]

BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron-electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel. [ABSTRACT FROM AUTHOR]

The outer membrane is a key virulence determinant of gram‐negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)6 enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail–LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis. [ABSTRACT FROM AUTHOR]

NaChBaC, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ~6 to ~4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment. [ABSTRACT FROM AUTHOR]

Coenzyme Q10 (CoQ10) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ10 was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A‐I. These particles, termed CoQ10 nanodisks (ND), contain 1.0 mg CoQ10/5 mg PtdCho/2 mg apoA‐I (97% CoQ10 solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ10 ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ10 into ND resulted in quenching of apoA‐I tryptophan fluorescence emission. Gel filtration chromatography of CoQ10 ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ10. Incubation of cultured cells with CoQ10 ND, but not empty ND, resulted in a significant increase in the CoQ10 content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ10 in lipid nanoparticles has been developed. The availability of CoQ10 ND provides a novel means to investigate biochemical aspects of CoQ10 uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise. [ABSTRACT FROM AUTHOR]

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cβ, C′) resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss. [ABSTRACT FROM AUTHOR]

Membranes proteins make up more than 60% of current drug targets and account for approximately 30% or more of the cellular proteome. Access to this important class of proteins has been difficult due to their inherent insolubility and tendency to aggregate in aqueous solutions. Understanding membrane protein structure and function demands novel means of membrane protein production that preserve both their native conformational state as well as function. Over the last decade, cell-free expression systems have emerged as an important complement to cell-based expression of membrane proteins due to their simple and customizable experimental parameters. One approach to overcome the solubility and stability limitations of purified membrane proteins is to support them in stable, native-like states within nanolipoprotein particles (NLPs), aka nanodiscs. This has become common practice to facilitate biochemical and biophysical characterization of proteins of interest. NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production for this purpose. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques to perform one-pot expression and self-assembly of membrane protein/NLP complexes. We describe how cell-free reactions can be modified to render control over nanoparticle size and monodispersity in support of membrane protein production. These modifications have been exploited to facilitate co-expression of full-length functional membrane proteins such as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In particular, we summarize the state of the art in NLP-assisted cell-free coexpression of these important classes of membrane proteins as well as evaluate the advances in and prospects for this technology that will drive drug discovery against these targets. We conclude with a prospective on the use of NLPs to produce as well as deliver functional mammalian membrane-bound proteins for a range of applications. [ABSTRACT FROM AUTHOR]

Recently, an enzymatic reaction was utilized to covalently link the N and C termini of membrane scaffold proteins to produce circularized nanodiscs that were more homogeneous and stable than standard nanodiscs. We continue this development and aim for obtaining high yields of stable and monodisperse nanodiscs for structural studies of membrane proteins by solution small‐angle scattering techniques. Based on the template MSP1E3D1, we designed an optimized membrane scaffold protein (His‐lsMSP1E3D1) with a sortase recognition motif and high abundance of solubility‐enhancing negative charges. With these modifications, we show that high protein expression is maintained and that the circularization reaction is efficient, such that we obtain a high yield of circularized membrane scaffold protein (csMSP1E3D1) and downstream circularized nanodiscs. We characterize the circularized protein and corresponding nanodiscs biophysically by small‐angle X‐ray scattering, size‐exclusion chromatography, circular dichroism spectroscopy, and light scattering and compare to noncircularized samples. First, we show that circularized and noncircularized (lsMSP1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that lsMSP1E3D1 nanodiscs are more stable compared to the template MSP1E3D1 nanodiscs as an effect of the extra negative charges and that csMSP1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in csMSP1E3D1 nanodiscs. Large‐scale production methods for circularized nanodiscs with improved thermal and temporal stability will facilitate better access to the nanodisc technology and enable applications at physiologically relevant temperatures. [ABSTRACT FROM AUTHOR]

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion. [ABSTRACT FROM AUTHOR]

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that accumulate around the circumference of a lipid bilayer. In this study, Pxt-5, which is an antimicrobial peptide isolated from the skin of Xenopus tropicalis, and its modified peptide (Modify- Pxt-5) were synthesized by solid-phase peptide synthesis (SPPS).Their surface properties, which are an important factor in inducing nanodisc formation, were investigated by circular dichroism (CD) spectroscopy, surface tension measurement, phospholipid vesicle clearance assay, and negative-staining transmission electron microscopy (NS-TEM). The α-helicity of Pxt-5 (8.4%) improved drastically to 45.6% by four amino-acid substitutions (Modify-Pxt-5). Both the peptides, having hydrophobic and hydrophilic faces, behaved like general surfactants, and the surface activity of Modify-Pxt-5 (CAC: 9.5x10-5 M, γCAC: 30.3 mN•m-1) was much higher than that of Pxt-5 (CAC: 7.9x10-5 M, γCAC: 38.1 mN•m-1). A turbid L-α- dimyristoylphosphatidylcholine (DMPC) vesicle solution (T = 0.3%) quickly turned transparent upon addition of Pxt-5 or Modify-Pxt-5. After twelve hours, the transmittance of vesicle solution with Modify-Pxt-5 (T = 96.2%) was found to be higher than that of vesicle solution with Pxt-5 (T = 83.5%), and then the micro-solubilized solutions were observed by NS-TEM. Interestingly, nanodisc structures were found in the vicinity of DMPC vesicles in both the images, and the average diameter of the nanodiscs was 11.2 ± 6.0 nm for those containing Pxt-5 and 10.8 ± 5.8 nm for those containing Modify-Pxt-5. It was also found that Modify-Pxt-5 effectively self-assembles into nanodiscs compared to Pxt-5 without any substitutions. [ABSTRACT FROM AUTHOR]

Cytochrome P450 17A1 (CYP17A1) catalyzes the synthesis of androgens from the steroid precursors pregnenolone and progesterone in a two‐step reaction process: allylic hydroxylation and carbo‐carbon bond scission. Cytochrome b5 (Cyt‐b5) is a stimulator of the second lyase reaction, but the chemical mechanism is unclear. We have shown previously that this stimulatory effect requires redox active Cyt‐b5. To investigate the origin of the lyase reaction enhancement by electron transfer from Cyt‐b5, we measured the reduction rates of oxy‐ferrous substrate‐bound CYP17A1 by Cyt‐b5 and by cytochrome P450 reductase (CPR) coincorporated in Nanodiscs using stopped flow spectroscopy. We observed that Cyt‐b5 reduces oxy‐ferrous CYP17A1 10‐fold faster than CPR, with the rate similar to that observed in a ternary complex of all three proteins. [ABSTRACT FROM AUTHOR]

The study of membrane proteins and receptors presents many challenges to researchers wishing to perform biophysical measurements to determine the structure, function, and mechanism of action of such components. In most cases, to be fully functional, proteins and receptors require the presence of a native phospholipid bilayer. In addition, many complex multiprotein assemblies involved in cellular communication require an integral membrane protein as well as a membrane surface for assembly and information transfer to soluble partners in a signaling cascade. Incorporation of membrane proteins into Nanodiscs renders the target soluble and provides a native bilayer environment with precisely controlled composition of lipids, cholesterol, and other components. Likewise, Nanodiscs provide a surface of defined area useful in revealing lipid specificity and affinities for the assembly of signaling complexes. In this review, we highlight several biophysical techniques made possible through the use of Nanodiscs. [ABSTRACT FROM AUTHOR]

Abstract: Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site‐specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell‐free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single‐subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N‐glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell‐free synthesized OSTs to glycosylate multiple target proteins with varying N‐glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane‐embedded OSTs from all kingdoms of life. [ABSTRACT FROM AUTHOR]

The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1. [ABSTRACT FROM AUTHOR]

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF).Understanding both the PA pore formation and LF translocation through the PA pore is crucial tomitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region. [ABSTRACT FROM AUTHOR]

Introduction: Integral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review. Areas covered: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing criticalin-vivoinformation. Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized. [ABSTRACT FROM PUBLISHER]

Historically, the main barrier to membrane protein investigations has been the tendency of membrane proteins to aggregate (due to their hydrophobic nature), in aqueous solution as well as on surfaces. The introduction of biomembrane mimetics has since stimulated momentum in the field. One such mimetic, the nanodisc (ND) system, has proved to be an exceptional system for solubilizing membrane proteins. Herein, we critically evaluate the advantages and imperfections of employing nanodiscs in biophysical and biochemical studies. Specifically, we examine the techniques that have been modified to study membrane proteins in nanodiscs. Techniques discussed here include fluorescence microscopy, solution-state/solid-state nuclear magnetic resonance, electron microscopy, small-angle X-ray scattering, and several mass spectroscopy methods. Newer techniques such as SPR, charge-sensitive optical detection, and scintillation proximity assays are also reviewed. Lastly, we cover how nanodiscs are advancing nanotechnology through nanoplasmonic biosensing, lipoprotein-nanoplatelets, and sortase-mediated labeling of nanodiscs. [ABSTRACT FROM AUTHOR]

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of smallmolecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids. [ABSTRACT FROM AUTHOR]

The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery. [Display omitted] • The ghrelin receptor binds sodium ions at its allosteric site • Sodium binding favors the receptor inactive/inactive-like conformational ensemble • Sodium binding decreases agonist affinity and receptor-catalyzed G protein activation Sodium is one of the most abundant ions in the body. Here, Ferré et al. provide evidence that this ion binds the ghrelin receptor and, by doing so, negatively impacts its signaling properties through an allosteric mechanism. As such, sodium could be a central player in ghrelin signaling. [ABSTRACT FROM AUTHOR]

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

Due to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly. [ABSTRACT FROM AUTHOR]

Summary Members of the YidC/Oxa1/Alb3 family universally facilitate membrane protein biogenesis, via mechanisms that have thus far remained unclear. Here, we investigated two crucial functional aspects: the interaction of YidC with ribosome:nascent chain complexes (RNCs) and the structural dynamics of RNC-bound YidC in nanodiscs. We observed that a fully exposed nascent transmembrane domain (TMD) is required for high-affinity YidC:RNC interactions, while weaker binding may already occur at earlier stages of translation. YidC efficiently catalyzed the membrane insertion of nascent TMDs in both fluid and gel phase membranes. Cryo-electron microscopy and fluorescence analysis revealed a conformational change in YidC upon nascent chain insertion: the essential TMDs 2 and 3 of YidC were tilted, while the amphipathic helix EH1 relocated into the hydrophobic core of the membrane. We suggest that EH1 serves as a mechanical lever, facilitating a coordinated movement of YidC TMDs to trigger the release of nascent chains into the membrane. [ABSTRACT FROM AUTHOR]

Complexin (Cpx) is thought to be a major regulator of soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE)-dependent membrane fusion. Although the inhibition of membrane fusion by Cpx has been frequently reported, its structural basis has been elusive and an anticipated disruption of the SNARE core has never been observed. In the present study, to mimic the natural environment, we assembled a single SNAREpin between two nanodisc membrane patches. Single-molecule FRET (smFRET) detects a large conformational change, specifically at the C-terminal half, whereas no conformational change is observed at the N-terminal half. Our results suggest that Cpx splits the C-terminal half of the SNARE core at least 10 Å (1 Å=0.1 nm), whereby inhibiting further progression of SNARE zippering and membrane fusion. [ABSTRACT FROM AUTHOR]

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly conserved set of membrane-associated proteins that mediate intracellular membrane fusion. Cognate SNAREs from two separate membranes zipper to facilitatemembrane apposition and fusion. Though the stable post-fusion conformation of SNARE complex has been extensively studied with biochemical and biophysical means, the pathway of SNARE zippering has been elusive. In this review, we describe some recent progress in understanding the pathway of SNARE zippering. We particularly focus on the half-zippered intermediate, which is most likely to serve as the main point of regulation by the auxiliary factors. [ABSTRACT FROM AUTHOR]

Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A-I (apoA-I) and phospholipids. Although peptide-based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA-I-based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline-punctuated bihelical amphipathic structure based on apoA-I mimetic peptides. NSP formed α-helical structure on 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA-I-POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA-I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396-10399, ). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales. [ABSTRACT FROM AUTHOR]

Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs. [ABSTRACT FROM AUTHOR]

A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general. [ABSTRACT FROM AUTHOR]

Neurotensin receptor type 1 (NTS1) is a G protein‐coupled receptor (GPCR) that affects cellular responses by initiating a cascade of interactions through G proteins. The kinetic details for these interactions are not well‐known. Here, NTS1‐nanodisc‐Gαs and Gαi1 interactions were studied. The binding affinities of Gαi1 and Gαs to NTS1 were directly measured by surface plasmon resonance (SPR) and determined to be 15 ± 6 nM and 31 ± 18 nM, respectively. This SPR configuration permits the kinetics of early events in signalling pathways to be explored and can be used to initiate descriptions of the GPCR interactome.Little is known of the kinetics of interactions between GPCRs and their signalling partners. NTS1 binds Gαi1 and Gαs with affinities of 15±6nM and 31±18nM (SE), respectively. This SPR assay may be applicable to multiple partners in the signalling cascade. We provide the first direct evidence for GPCR‐G protein coupling in nanodiscs. [ABSTRACT FROM AUTHOR]

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up awide range of possibilities for the future study of their structure and function. [ABSTRACT FROM AUTHOR]

Membrane proteins (MPs) are of central interest for the pharmaceutical industry but their production is usually a challenging task. The complex folding mechanisms of newly synthesized MPs often require interactions with specific compounds for improved stability. Conditions for the production of high-quality samples are therefore difficult to predict and frequently cannot be provided in conventional protein expression platforms. Cell-free (CF) biosynthetic systems allow, in contrast to living cells, nonrestricted access to the protein production machinery. Reaction conditions can be adjusted according to particular requirements and modified by supplementing single additives or even cocktails of compounds. These options have initiated completely new research fields for the cotranslational stabilization and folding of MPs in artificial environments. Based on established and efficient CF production protocols, a recent focus was to explore and define suitable supplements for CF expression reactions that are useful for the generation of high-quality MP samples. Besides classical detergents and lipids, a variety of new compounds with interesting properties have been discovered and synthesized. We compile the currently available toolbox for MP solubilization in CF systems and summarize new developments and perspectives for the directed modulation of CF biosynthetic environments. [ABSTRACT FROM AUTHOR]

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment. [ABSTRACT FROM AUTHOR]

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized, pre-fusion conformations have been elusive. We used single-molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. Although the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure, the membrane-proximal Cterminal half shows high FRET, representing a helical bundle (45%), low FRET, reflecting a frayed conformation (39%) or mid FRET revealing an as-yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1. [ABSTRACT FROM AUTHOR]

Abstract Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally through their regulated affinities for extracellular ligands and intracellular signaling molecules. Such bidirectional signaling by integrins is enabled by the conformational changes that are often linked among extracellular, transmembrane and cytoplasmic domains. Here, we review how talin-integrin and kindlin-integrin interactions, in cooperation with talin-lipid and kindlin-lipid interactions, regulate integrin affinities and how the progress in these areas helps us understand integrin-related diseases. [ABSTRACT FROM AUTHOR]

Within the last decade, nanoscale lipid bilayers have emerged as powerful experimental systems in the analysis of membrane proteins (MPs) for both basic and applied research. These discoidal lipid lamellae are stabilized by annuli of specially engineered amphipathic polypeptides (nanodiscs) or polymers (SMALPs/Lipodisqs®). As biomembrane mimetics, they are well suited for the reconstitution of MPs within a controlled lipid environment. Moreover, because they are water-soluble, they are amenable to solution-based biochemical and biophysical experimentation. Hence, due to their solubility, size, stability, and monodispersity, nanoscale lipid bilayers offer technical advantages over more traditional MP analytic approaches such as detergent solubilization and reconstitution into lipid vesicles. In this article, we review some of the most recent advances in the synthesis of polypeptide- and polymer-bound nanoscale lipid bilayers and their application in the study of MP structure and function. [ABSTRACT FROM AUTHOR]