723 results on '"Nanodiscs"'
Search Results
2. The effect of lipid composition on the thermal stability of nanodiscs
- Author
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Knetsch, Tim G.J. and Ubbink, Marcellus
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- 2024
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3. ABC transporter activity is affected by the size of lipid nanodiscs.
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Nouel Barreto, Annabella, Cuello, Luis G., and Zoghbi, Maria E.
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SCAFFOLD proteins , *MEMBRANE proteins , *ADENOSINE triphosphatase , *LIPOSOMES , *LIPIDS - Abstract
Lipid nanodiscs have become a widely used approach for studying membrane proteins thanks to several advantages they offer. They have been especially useful for studying ABC transporters, despite the growing concern about the possible restriction of the conformational changes of the transporters due to the small size of the discs. Here, we performed a systematic study to determine the effect of the nanodisc size on the ATPase activity of model ABC transporters from human, plant, and bacteria. Our data confirm that the activity of the transporters and their response to regulatory molecules is affected by the nanodisc size. Our findings suggest the use of larger membrane scaffold proteins (MSPs), such as MSP2N2 nanodiscs, to minimize alterations caused by the commonly used small MSP1D1. [ABSTRACT FROM AUTHOR]
- Published
- 2025
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4. The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling
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Elise S Bruguera, Jacob P Mahoney, and William I Weis
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Wnt ,cell signaling ,specificity ,co-receptor ,nanodiscs ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.
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- 2025
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5. Exploring GPCR conformational dynamics using single-molecule fluorescence.
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Agyemang, Eugene, Gonneville, Alyssa N., Tiruvadi-Krishnan, Sriram, and Lamichhane, Rajan
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G protein coupled receptors , *X-ray microscopy , *FLUORESCENCE microscopy , *STRUCTURAL dynamics , *X-ray crystallography , *ELECTRON microscopy - Abstract
[Display omitted] • Explores different approaches for expressing, purifying, and labeling GPCRs both in vitro and in live cell environments. • Discusses specific sample preparation methods for single-molecule fluorescence microscopy. • Examines the unique challenges involved in each stage of sample preparation for single-molecule GPCR experiments. • Highlights recent applications of single-molecule TIRF microscopy in investigating different classes of human GPCRs. G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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6. Reconstitution and characterization of BRAF in complex with 14‐3‐3 and KRAS4B on nanodiscs.
- Author
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Liu, Ningdi F., Enomoto, Masahiro, Marshall, Christopher B., and Ikura, Mitsuhiko
- Abstract
RAF kinases are key components of the RAS‐MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto‐inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14‐3‐3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14‐3‐3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP‐dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full‐length BRAF:14‐3‐3 complexes for KRAS4B‐conjugated nanodiscs (RAS‐ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2‐Dioleoyl‐sn‐glycero‐3‐phospho‐L‐serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14‐3‐3 with RAS‐ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14‐3‐3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS‐ND increased activity of both states of BRAF. The reconstituted assembly of full‐length BRAF with 14‐3‐3 and KRAS on a cell‐free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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7. Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.
- Author
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Babot, Marion, Boulard, Yves, Agouda, Samira, Pieri, Laura, Fieulaine, Sonia, Bressanelli, Stéphane, and Gervais, Virginie
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SARS-CoV-2 , *MEMBRANE proteins , *TRANSMEMBRANE domains , *PHOSPHOLIPIDS - Abstract
As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted puri fied nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negativestain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFoldpredicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies. [ABSTRACT FROM AUTHOR]
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- 2024
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8. Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation.
- Author
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Imura, Tomohiro, Yanagisawa, Satohiro, Ikeda, Yuri, Moriyama, Ryodai, Sakai, Kenichi, Sakai, Hideki, and Taira, Toshiaki
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SURFACTIN , *MEMBRANE transport proteins , *SOLUBILIZATION , *MOLECULAR recognition , *TRANSMISSION electron microscopy , *UBIQUINONES , *LIPIDS - Abstract
Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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9. Encapsulation of propolis extracts in aqueous formulations by using nanovesicles of lipid and poly(styrene-alt-maleic acid)
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Chatmani Buachi, Charothar Thammachai, Brian J. Tighe, Paul D. Topham, Robert Molloy, and Patchara Punyamoonwongsa
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Nanodiscs ,biomimetic ,drug delivery ,propolis ,styrene maleic acid ,nanoencapsulation ,Biotechnology ,TP248.13-248.65 ,Medical technology ,R855-855.5 - Abstract
AbstractBee propolis has been used in alternative medicine to treat various diseases. Due to its limited water solubility, it is often used in combination with alcohol solvents, causing skin irritation and immune response. To solve this, the new drug delivery system, based on the lipid nanodiscs of 1,2-dimyristoyl-sn-glycero-3-phosphochline (DMPC) and poly(styrene-alt-maleic acid) (PSMA), were created in an aqueous media. At the excess polymer concentrations, the PSMA/DMPC complexation produced the very fine nanoparticles (18 nm). With the increased molar ratio of styrene to maleic acid (St/MA) in the copolymer structure, the lipid nanodisc showed the improved encapsulation efficiency (EE%), comparing to their corresponding aqueous formulations. The maximum value had reached to around 20% when using the 2:1 PSMA precursor. Based on the cytotoxicity test, these nanoparticles were considered to be non-toxic over the low dose administration region (
- Published
- 2023
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10. Bioelectronic Tongue for Identifying and Masking Bitterness Based on Bitter Taste Receptor Agonism and Antagonism.
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Hwang, Jun Young, Kim, Kyung Ho, Seo, Sung Eun, Nam, Youngju, Jwa, Sanghee, Yang, Inwoo, Park, Tai Hyun, Kwon, Oh Seok, and Lee, Seung Hwan
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BITTERNESS (Taste) , *TASTE receptors , *TONGUE , *PATIENT compliance , *FIELD-effect transistors , *ESCHERICHIA coli - Abstract
Bitterness elicits unpleasant sensations in humans, which can hinder the acceptance of foods and medication adherence. Therefore, identifying and masking bitter tastes is crucial for developing palatable foods and promoting medication compliance in the food and pharmaceutical industries. To achieve this, employing agonism and antagonism of bitter taste receptors as effective strategies at the molecular level is essential. In this study, a bioelectronic tongue is developed to characterize the agonism and antagonism of bitter taste receptors. The human bitter taste receptors hTAS2R16 and hTAS2R31 are produced using an Escherichia coli expression system and reconstituted into nanodiscs (NDs). Subsequently, hTAS2R16‐ and hTAS2R31‐NDs are immobilized on the surface of graphene field‐effect transistors (FETs) to construct bioelectronic tongues. The developed system sensitively detected the bitter agonists, salicin and saccharin, at concentrations as low as 100 fM, with high selectivity in real‐time. The dose‐dependent curves shifted and K values decreased by the antagonists of hTAS2R16 and hTAS2R31, indicating antagonism‐based masking of bitter taste. Therefore, the developed bioelectronic tongue holds promise for identifying bitter tastes and evaluating the masking of bitterness based on the agonism and antagonism of hTAS2Rs. [ABSTRACT FROM AUTHOR]
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- 2023
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11. Phospholipids impact the protective effects of HDL-mimetic nanodiscs against lipopolysaccharide-induced inflammation.
- Author
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Kim, Sang Yeop, Kang, Jukyung, Fawaz, Maria V, Yu, Minzhi, Xia, Ziyun, Morin, Emily E, Mei, Ling, Olsen, Karl, Li, Xiang-An, and Schwendeman, Anna
- Abstract
Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo, suggesting a potential sepsis treatment. Sepsis is triggered by endotoxins released by bacteria. These endotoxins trigger an exaggerated inflammatory response, leading to widespread inflammation and organ damage. Synthetic high-density lipoprotein (sHDL) is a potential treatment of sepsis by neutralizing endotoxins and regulating inflammatory responses. The phospholipid components of sHDL may affect the effectiveness of sHDL against sepsis. In this study, we prepared sHDLs with different phospholipids and compared their anti-septic effects on cells and in animal models. We found that sHDL made from DMPC presented the best anti-septic effects, possibly because DMPC-sHDL had the best fluidity at body temperature. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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12. Translational Challenges and Prospective Solutions in the Implementation of Biomimetic Delivery Systems.
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Wang, Zhe, Wang, Xinpei, Xu, Wanting, Li, Yongxiao, Lai, Ruizhi, Qiu, Xiaohui, Chen, Xu, Chen, Zhidong, Mi, Bobin, Wu, Meiying, and Wang, Junqing
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NANOMEDICINE , *BIOLOGICAL systems , *EXTRACELLULAR vesicles , *INDIVIDUALIZED medicine , *LIPOSOMES , *VIRUS-like particles - Abstract
Biomimetic delivery systems (BDSs), inspired by the intricate designs of biological systems, have emerged as a groundbreaking paradigm in nanomedicine, offering unparalleled advantages in therapeutic delivery. These systems, encompassing platforms such as liposomes, protein-based nanoparticles, extracellular vesicles, and polysaccharides, are lauded for their targeted delivery, minimized side effects, and enhanced therapeutic outcomes. However, the translation of BDSs from research settings to clinical applications is fraught with challenges, including reproducibility concerns, physiological stability, and rigorous efficacy and safety evaluations. Furthermore, the innovative nature of BDSs demands the reevaluation and evolution of existing regulatory and ethical frameworks. This review provides an overview of BDSs and delves into the multifaceted translational challenges and present emerging solutions, underscored by real-world case studies. Emphasizing the potential of BDSs to redefine healthcare, we advocate for sustained interdisciplinary collaboration and research. As our understanding of biological systems deepens, the future of BDSs in clinical translation appears promising, with a focus on personalized medicine and refined patient-specific delivery systems. [ABSTRACT FROM AUTHOR]
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- 2023
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13. Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis.
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Vénien-Bryan, Catherine and Fernandes, Carlos A. H.
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MEMBRANE proteins , *ELECTRON microscopy , *MICROSCOPY , *BANKING industry , *PROTEIN structure , *VITRIFICATION - Abstract
Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
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14. What's the defect? Using mass defects to study oligomerization of membrane proteins and peptides in nanodiscs with native mass spectrometry.
- Author
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Townsend, Julia A. and Marty, Michael T.
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MEMBRANE proteins , *MASS spectrometry , *ANTIMICROBIAL peptides , *PEPTIDES , *OLIGOMERIZATION , *MEMBRANE lipids , *AMYLOID - Abstract
[Display omitted] • Combining native mass spectrometry and nanodiscs reveals the oligomeric states of membrane proteins and peptides inside lipid bilayers. • Mass defect analysis can be used to better understand the stoichiometry and lipid preferences of membrane proteins and peptides. • This review describes how to perform macromolecular mass defect analysis, limitations of the technique, strategies for interpretation, and applications to studying the membrane biophysics of antimicrobial peptides, viroporins, and amyloid proteins. Many membrane proteins form functional complexes that are either homo- or hetero-oligomeric. However, it is challenging to characterize membrane protein oligomerization in intact lipid bilayers, especially for polydisperse mixtures. Native mass spectrometry of membrane proteins and peptides inserted in lipid nanodiscs provides a unique method to study the oligomeric state distribution and lipid preferences of oligomeric assemblies. To interpret these complex spectra, we developed novel data analysis methods using macromolecular mass defect analysis. Here, we provide an overview of how mass defect analysis can be used to study oligomerization in nanodiscs, discuss potential limitations in interpretation, and explore strategies to resolve these ambiguities. Finally, we review recent work applying this technique to studying formation of antimicrobial peptide, amyloid protein, and viroporin complexes with lipid membranes. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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15. Advances in nanodisc platforms for membrane protein purification.
- Author
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Pettersen, John M., Yang, Yaxin, and Robinson, Anne S.
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MEMBRANE proteins , *SCAFFOLD proteins , *MALEIC acid , *CELL membranes , *POLYMETHACRYLATES , *ELECTRON microscopy , *POLYMERS - Abstract
Nanodiscs have become an extremely valuable tool for membrane protein (MP) research. The first nanodiscs, membrane scaffold protein nanodiscs (MSPNDs), have seen substantial technological development over the past decade, and salipros and copolymer nanodiscs have recently emerged as promising alternatives to MSPNDs. Recent advances include the introduction of circular MSPNDs, improvements to robust nanodisc method development, and increased breakthrough applications for MP structures and biophysical binding studies. Salipros are able to adapt to the size of different MPs by using a single scaffold variant. Although very recently established, salipro methodology and applications have seen rapid development. Copolymer nanodiscs can incorporate MPs in their endogenous lipids via extracting the MP directly from the cell membranes. Three groups of copolymers – styrene–maleic acid (SMA), diisobutylene maleic acid (DIBMA), and polymethacrylate polymer (PMA) – have been employed with varying success to stabilize MPs in an active state. Membrane scaffold protein nanodiscs (MSPNDs) are an invaluable tool for improving purified membrane protein (MP) stability and activity compared to traditional micellar methods, thus enabling an increase in high-resolution MP structures, particularly in concert with cryogenic electron microscopy (cryo-EM) approaches. In this review we highlight recent advances and breakthroughs in MSPND methodology and applications. We also introduce and discuss saposin–lipoprotein nanoparticles (salipros) and copolymer nanodiscs which have recently emerged as authentic MSPND alternatives. We compare the advantages and disadvantages of MSPNDs, salipros, and copolymer nanodisc technologies to highlight potential opportunities for using each platform for MP purification and characterization. [ABSTRACT FROM AUTHOR]
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- 2023
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16. Engineering Substrate-Mediated Localized Surface Plasmons in Gold Nanodiscs.
- Author
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Chirumamilla, Anisha, Salazar, Maria H., Wang, Deyong, Kristensen, Peter K., Sutherland, Duncan S., Chirumamilla, Manohar, Popok, Vladimir N., and Pedersen, Kjeld
- Subjects
SURFACE plasmons ,FLUX pinning ,SURFACE plasmon resonance ,SERS spectroscopy ,OPTICAL measurements ,GAS flow - Abstract
A variety of nanostructures capable of generating strong local electromagnetic fields (hot spots) in interaction with radiation have been under intensive investigation towards plasmonic applications in surface-enhanced Raman scattering (SERS), biosensing, broadband absorbers, thermophotovoltaics, photocatalysis, etc. In many cases, these nanostructures are formed on a surface or embedded into a near-surface layer of the dielectric substrate, making some part of the field dissipate into the bulk and not contribute to the desired plasmonic functionality. To reduce such losses, the interface between the metallic nanostructures and the dielectric environment should be engineered. In the current work, Au nanodiscs are fabricated on Si posts of very small diameter (pin-shaped structures), enabling them to decouple the strong optical near fields localized at the nanodiscs from the bulk Si substrate. The Si post diameter is optimized by adjusting the gas flow rates in reactive-ion etching, resulting in a minimum post diameter of 20 nm at the nanodisc interface. The effect of this diameter on the localized surface plasmon resonance of the nanodisc is investigated with linear optical spectroscopic measurements, where a significant spectral blue shift of the resonance band is noticed compared to similar discs formed on bulk Si surface. The experimental results are compared with modelling where a 3-fold increase in the electric field enhancement is demonstrated. The fabricated pin-shaped nanostructures are tested in SERS measurements showing a significant increase in the enhancement factor in the order of 10
6 . Thus, this work suggests a way of engineering 3D morphology to tune the substrate influence on the plasmonic properties of nanostructures and to develop efficient nanofabrication technologies. [ABSTRACT FROM AUTHOR]- Published
- 2023
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17. Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System.
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Rouchidane Eyitayo, Akandé, Boudier-Lemosquet, Axel, Chaignepain, Stéphane, Priault, Muriel, and Manon, Stéphen
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PROTEIN synthesis , *ESCHERICHIA coli , *C-terminal residues , *MITOCHONDRIAL membranes , *CELL survival - Abstract
The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
18. Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs.
- Author
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Surya, Wahyu, Yong, Clare Pei Yii, Tyagi, Anu, Bhushan, Shashi, and Torres, Jaume
- Subjects
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ESCHERICHIA coli , *MEMBRANE proteins , *OLIGOMERIZATION , *SCAFFOLD proteins , *DETERGENTS , *AQUAPORINS - Abstract
Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
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19. Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation
- Author
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Tomohiro Imura, Satohiro Yanagisawa, Yuri Ikeda, Ryodai Moriyama, Kenichi Sakai, Hideki Sakai, and Toshiaki Taira
- Subjects
surfactin ,phospholipid ,nanodiscs ,fibrous aggregates ,Organic chemistry ,QD241-441 - Abstract
Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.
- Published
- 2024
- Full Text
- View/download PDF
20. Lipid nanodiscs of poly(styrene-alt-maleic acid) to enhance plant antioxidant extraction
- Author
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Punyamoonwongsa Patchara
- Subjects
extraction ,membrane lysis ,styrene maleic acid ,antioxidants ,nanodiscs ,Polymers and polymer manufacture ,TP1080-1185 - Abstract
Plant antioxidants can be applied in the management of various human diseases. Despite these, extraction of these compounds still suffers from residual solvent impurities, low recovery yields, and the risks of undesirable chemical changes. Inspired by the protein–lipid interactions in the cell membranes, we proposed using poly(styrene-alt-maleic acid) (PSMA) to destabilize and associate with the bilayer lipids into the membrane-like nanodiscs. Such nanostructures could serve as protective reservoirs for the active compounds to reside with preserved bioactivities. This concept was demonstrated in the antioxidant extraction from robusta coffee leaves. Results indicated that aqueous PSMA extraction (no buffer agent) yielded products with the highest contents of phenolic acids (11.6 mg GAE·g−1) and flavonoids (9.6 mg CE·g−1). They also showed the highest antioxidant activity (IC50 = 3.7 µg·mL−1) compared to those obtained by typical sodium dodecyl sulfate and water extraction. This biomimetic approach could be considered for developing environmentally friendly extraction protocols in the future.
- Published
- 2022
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21. The Self‐Association of the KRAS4b Protein is Altered by Lipid‐Bilayer Composition and Electrostatics.
- Author
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Lee, Ki‐Young, Ikura, Mitsuhiko, and Marshall, Christopher B.
- Subjects
- *
ELECTROSTATICS , *MEMBRANE lipids , *MEMBRANE proteins , *PHOSPHATIDYLSERINES , *BILAYER lipid membranes - Abstract
KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self‐association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self‐assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self‐association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5‐α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge‐reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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22. A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes.
- Author
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Sawczyc, Henry, Heit, Sabine, and Watts, Anthony
- Subjects
- *
NANOPARTICLES , *LIGHT absorbance , *NANOPARTICLE size , *PHOTOMETRY , *LIGHT scattering , *POLYMETHACRYLATES , *EFFECT of salt on plants - Abstract
From the discovery of the first membrane-interacting polymer, styrene maleic-acid (SMA), there has been a rapid development of membrane solubilising polymers. These new polymers can solubilise membranes under a wide range of conditions and produce varied sizes of nanoparticles, yet there has been a lack of broad comparison between the common polymer types and solubilising conditions. Here, we present a comparative study on the three most common commercial polymers: SMA 3:1, SMA 2:1, and DIBMA. Additionally, this work presents, for the first time, a comparative characterisation of polymethacrylate copolymer (PMA). Absorbance and dynamic light scattering measurements were used to evaluate solubilisation across key buffer conditions in a simple, adaptable assay format that looked at pH, salinity, and divalent cation concentration. Lipid-polymer nanoparticles formed from SMA variants were found to be the most susceptible to buffer effects, with nanoparticles from either zwitterionic DMPC or POPC:POPG (3:1) bilayers only forming in low to moderate salinity (< 600 mM NaCl) and above pH 6. DIBMA-lipid nanoparticles could be formed above a pH of 5 and were stable in up to 4 M NaCl. Similarly, PMA-lipid nanoparticles were stable in all NaCl concentrations tested (up to 4 M) and a broad pH range (3–10). However, for both DIBMA and PMA nanoparticles there is a severe penalty observed for bilayer solubilisation in non-optimal conditions or when using a charged membrane. Additionally, lipid fluidity of the DMPC-polymer nanoparticles was analysed through cw-EPR, showing no cooperative gel-fluid transition as would be expected for native-like lipid membranes. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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23. Tuning the surface morphologies of ZnO nanofilms for enhanced sensitivity and selectivity of CO2 gas sensor.
- Author
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Taha, T. A., Saad, Rana, Zayed, Mohamed, Shaban, Mohamed, and Ahmed, Ashour M.
- Subjects
- *
ZINC oxide films , *GAS detectors , *NANOFILMS , *SURFACE morphology , *ZINC oxide thin films , *FIELD emission electron microscopy , *CHEMICAL solution deposition - Abstract
In this work, ZnO has been synthesized with a variety of nanomorphologies, including nanorods (NRs), nanodiscs (NDs), and nanorods/nanodiscs (NRs/NDs), to enhance CO2 gas detection at room temperature. The ZnO nanostructures were made by combining the successive ionic layer adsorption and reaction (SILAR) strategy and the chemical bath deposition (CBD) method. The time of CBD varied from 6 to 12 h. Several techniques, including X-ray diffraction (XRD) spectroscopy, energy-dispersive X-ray (EDAX) spectrometry, optical spectrophotometer, and field emission scanning electron microscopy (FE-SEM), were used to investigate the manufactured ZnO nanostructures. The FE-SEM demonstrates that by increasing the deposition period of CBD from 6 to 12 h, the shape of ZnO nanostructures changed from NRs/NDs to NDs. According to the XRD, all ZnO nanostructured samples exhibit hexagonal wurtzite structures with (002) preferred orientation. Additionally, the crystallite size along orientation (002) increases from 63 to 65 nm as the deposition duration increases from 6 to 12 h. The bandgap of ZnO was reduced from 3.62 to 3.31 eV. When the deposition time is increased from 6 to 12 h, the sensitivity increases from 8.46 to 28.7%, the detection limit rises from 4.65 to 9.95 SCCM, and the limit of quantification rises from 15.52 to 33.16 SCCM. Moreover, the ZnO @ 12 h sensors has excellent selectivity as well since it reacts to CO2 with a higher response sensitivity than it does to other gases like hydrogen and ammonia. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
24. Development of a saposin A based native-like phospholipid bilayer system for NMR studies
- Author
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Chien, Chih-Ta and Nietlispach, Daniel
- Subjects
572 ,NMR spectroscopy ,Nanodiscs ,Membrane protein ,GPCR ,Saposin lipid nanoparticles - Abstract
Membrane proteins are important targets that represent more than 50% of current drug targets. However, characterisation of membrane proteins falls behind compared to their soluble counterparts. The most challenging part of membrane protein research is finding a suitable membrane mimetic that stabilises them in solution and maintains their native structure and function. The recently developed saposin-A (SapA) based lipid nanoparticle system seems to be advantageous over existing membrane mimetic system. It provides a native-like lipid bilayer, high incorporation yield and more importantly size adaptability. SapA lipid nanoparticles have been applied to structural studies and two high-resolution structures of membrane proteins were previously obtained using cryo-electron microscopy. This thesis aimed to study small-to-medium sized membrane proteins in SapA lipid nanoparticles using NMR spectroscopy. We first explore the mechanism of SapA lipid nanoparticle formation for the purpose of establishing an incorporation protocol that can be applied to most membrane proteins. The effect of pH and the presence of detergents on the opening of SapA was investigated in Chapter 2. A proposed energy diagram describing the mechanism of SapA opening is reported with which we were able to develop a protocol that can generate different sizes of SapA-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) nanoparticles. In addition, we also showed that SapA can form lipid nanoparticles with various lipid compositions, showing the versatility of the system. In Chapter 3, we validated the ability of SapA lipid nanoparticles to be used as a membrane mimetic. A -barrel model protein, bacterial outer membrane protein X (OmpX), was incorporated into SapA-DMPC nanoparticles and a 2D 15N-1H correlation NMR spectrum was recorded. Our result was compared to the NMR parameters of the same protein in MSP nanodiscs from the literature, and it was concluded that SapA lipid nanoparticles indeed provide a lipid bilayer environment similar to MSP nanodiscs. Because of high incorporation yield, we were able to incorporate OmpX into different lipid compositions to investigate the effect of lipid head groups and aliphatic chains on the membrane protein's chemical environment. Next, the applicability of SapA lipid nanoparticles was expanded to -helical transmembrane proteins in Chapter 4. Two microbial rhodopsins, Anabaena sensory rhodopsin (ASR) and Natronomonas pharaonis sensory rhodopsin II (pSRII), were tested. The parameters for expression and purification of ASR were first screened for the optimal yield. Although incorporation of ASR resulted in inhomogeneous particles due to imperfect experimental procedure, pSRII in SapA-DMPC nanoparticles showed high sample quality. The 2D NMR spectrum of pSRII in SapA-DMPC nanoparticles shows distinct differences to pSRII in detergent micelles, suggesting substantial effects from the membrane mimetic on the conformation of the membrane protein. Despite the good NMR spectral quality considering the large particle size, perdeuteration of pSRII and the lipids will be necessary for further investigation. With the SapA lipid nanoparticles established, we aimed to use it for the study of a biologically important G protein-coupled receptor, 1-adrenergic receptor (1AR), discussed in Chapter 5. The possibility of expressing 1AR using a cell-free expression system was explored first. Although a good amount of the protein was obtained, only a fraction of it was functional. Therefore, a conventional baculovirus-insect cell expression system was used to produce selective isotope labelled 1AR for NMR studies. NMR spectra of 1AR in SapA-DMPC nanoparticles with activating ligands and an intracellular binding partner were recorded and compared to the spectra of the same protein in detergents. This revealed a more active-like conformation of ligand-bound 1AR in the lipid bilayer, suggesting that certain parts of the protein are sensitive to the membrane mimetic used. This emphasises the importance of using a native-like membrane mimetic to capture the full properties of membrane proteins. In conclusion, I demonstrate in this thesis that SapA lipid nanoparticles are a versatile membrane mimetic system that can accommodate membrane proteins with different sizes and folds. This system is also compatible with solution NMR spectroscopy enabling structure and dynamics studies of biologically important membrane proteins. We believe SapA lipid nanoparticles will have a significant impact on membrane protein research in the future.
- Published
- 2019
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25. On the structure and function of multidrug efflux pumps
- Author
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Neuberger, Arthur, van Veen, Hendrik W., and Luisi, Ben F.
- Subjects
616.9 ,cryo-electron microscopy ,membrane proteins ,antibiotic resistance ,multidrug efflux pumps ,multidrug resistance ,structural biology ,nanodiscs ,lipids ,drug efflux ,cardiolipin ,transport assays ,Hoechst - Abstract
Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter's functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the 'plasticity' in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The 'cryo-EM revolution' has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6.
- Published
- 2019
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26. Detergents and alternatives in cryo-EM studies of membrane proteins
- Author
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Li Shuo
- Subjects
detergent ,micelle ,nanodiscs ,cryo-EM ,membrane protein ,Biochemistry ,QD415-436 ,Genetics ,QH426-470 - Abstract
Structure determination of membrane proteins has been a long-standing challenge to understand the molecular basis of life processes. Detergents are widely used to study the structure and function of membrane proteins by various experimental methods, and the application of membrane mimetics is also a prevalent trend in the field of cryo-EM analysis. This review focuses on the widely-used detergents and corresponding properties and structures, and also discusses the growing interests in membrane mimetic systems used in cryo-EM studies, providing insights into the role of detergent alternatives in structure determination.
- Published
- 2022
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27. Binding of yeast and human cytochrome c to cardiolipin nanodiscs at physiological ionic strength.
- Author
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Frederick, Ariel K. and Bowler, Bruce E.
- Subjects
- *
BINDING site assay , *GEL permeation chromatography , *SURFACE potential , *CARDIOLIPIN , *BINDING sites - Abstract
Binding of cytochrome c (Cyt c) to membranes containing cardiolipin (CL) is of considerable interest because of the importance of this interaction in the early stages of apoptosis. The molecular-level determinants of this interaction are still not well defined and there appear to be species-specific differences in Cyt c affinity for CL-containing membranes. Many studies are carried out at low ionic strength far from the 100–150 mM ionic strength within mitochondria. Similarly, most binding studies are done at Cyt c concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cyt c present in mitochondria. In this study, we evaluate binding of human and yeast Cyt c to CL nanodiscs using size exclusion chromatography at 25 μM Cyt c concentration and 100 mM ionic strength. We find that yeast Cyt c affinity for CL nanodiscs is much stronger than that of human Cyt c. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cyt c binding to CL nanodiscs than lysines 72 and 73, counter to results at lower ionic strength. Analysis of the electrostatic surface potential of human versus yeast Cyt c shows that the positive potential due to lysines 86 and 87 and other nearby lysines (4, 5, 11, 89) is stronger than that due to lysines 72 and 73. In the case of human Cyt c the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A. Yeast iso-1-cytochrome c binds more strongly to cardiolipin nanodiscs than human cytochrome c at physiological ionic strength because the charge distribution near site A (lysines 72, 73, 86, 87) is more uniformly positive. Mutagenesis studies on site A lysines suggest that N- and C-terminal helix lysines contribute to electrostatic binding. [Display omitted] • Human cytochrome c does not bind to cardiolipin nanodiscs at 100 mM ionic strength. • Yeast iso-1-cytochrome c binds to cardiolipin nanodiscs at 100 mM ionic strength. • The electrostatic surface potential is stronger for yeast versus human cytochrome c. • Site A lysines 86 and 87 are most important for binding at 100 mM ionic strength. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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28. Translational Challenges and Prospective Solutions in the Implementation of Biomimetic Delivery Systems
- Author
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Zhe Wang, Xinpei Wang, Wanting Xu, Yongxiao Li, Ruizhi Lai, Xiaohui Qiu, Xu Chen, Zhidong Chen, Bobin Mi, Meiying Wu, and Junqing Wang
- Subjects
biomimetic ,bioinspired ,nanodiscs ,liposomes ,virus-like particles ,albumin ,Pharmacy and materia medica ,RS1-441 - Abstract
Biomimetic delivery systems (BDSs), inspired by the intricate designs of biological systems, have emerged as a groundbreaking paradigm in nanomedicine, offering unparalleled advantages in therapeutic delivery. These systems, encompassing platforms such as liposomes, protein-based nanoparticles, extracellular vesicles, and polysaccharides, are lauded for their targeted delivery, minimized side effects, and enhanced therapeutic outcomes. However, the translation of BDSs from research settings to clinical applications is fraught with challenges, including reproducibility concerns, physiological stability, and rigorous efficacy and safety evaluations. Furthermore, the innovative nature of BDSs demands the reevaluation and evolution of existing regulatory and ethical frameworks. This review provides an overview of BDSs and delves into the multifaceted translational challenges and present emerging solutions, underscored by real-world case studies. Emphasizing the potential of BDSs to redefine healthcare, we advocate for sustained interdisciplinary collaboration and research. As our understanding of biological systems deepens, the future of BDSs in clinical translation appears promising, with a focus on personalized medicine and refined patient-specific delivery systems.
- Published
- 2023
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- View/download PDF
29. Micro-ellipsometry of square lattices of plasmonic nanodiscs on dielectric substrates and in metal-insulator-metal configurations
- Author
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Eugene Bortchagovsky, P. Christian Simo, Ilya Milekhin, Jia Tang, Dietrich R.T. Zahn, and Monika Fleischer
- Subjects
Micro-ellipsometry ,Nanodiscs ,Square lattices ,Dark-field spectra ,Metal-insulator-metal structures ,Electronics ,TK7800-8360 ,Technology (General) ,T1-995 - Abstract
This article demonstrates the capability of imaging micro-ellipsometry in the investigation of ordered plasmonic nanostructures on different substrates. The difference of the resonances of similar square lattices of plasmonic nanoparticles on a dielectric substrate and in a metal-insulator-metal configuration is analyzed. The influence of the surface plasmon on interparticle interactions becomes clearly apparent. Comparison of the obtained data with dark-field scattering spectra proves the consistency of the measured results as well as the complementary information received from both methods.
- Published
- 2023
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30. DNA-nanostructure-templated assembly of planar and curved lipid-bilayer membranes
- Author
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Mostafa A. Elbahnasawy and Mahmoud L. Nasr
- Subjects
DNA nanostructures ,liposomes ,DNA-corralled nanodiscs ,DNA origami ,nanodiscs ,membrane proteins ,Chemistry ,QD1-999 - Abstract
Lipid-bilayer nanodiscs and liposomes have been developed to stabilize membrane proteins in order to study their structures and functions. Nanodiscs are detergent-free, water-soluble, and size-controlled planar phospholipid-bilayer platforms. On the other hand, liposomes are curved phospholipid-bilayer spheres with an aqueous core used as drug delivery systems and model membrane platforms for studying cellular activities. A long-standing challenge is the generation of a homogenous and monodispersed lipid-bilayer system with a very wide range of dimensions and curvatures (elongation, bending, and twisting). A DNA-origami template provides a way to control the shapes, sizes, and arrangements of lipid bilayers via enforcing the assembly of lipid bilayers within the cavities created by DNA nanostructures. Here, we provide a concise overview and discuss how to design planar and curved lipid-bilayer membranes by using DNA-origami nanostructures as templates. Finally, we will discuss the potential applications of DNA-origami nanostructures in the structural and functional studies of large membrane proteins and their complexes.
- Published
- 2023
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31. Polymer-Nanodiscs as a Novel Alignment Medium for High-Resolution NMR-Based Structural Studies of Nucleic Acids.
- Author
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Krishnarjuna, Bankala, Ravula, Thirupathi, Faison, Edgar M., Tonelli, Marco, Zhang, Qi, and Ramamoorthy, Ayyalusamy
- Subjects
- *
SMALL molecules , *POLYZWITTERIONS , *X-ray crystallography , *BIOMOLECULES , *NUCLEIC acids , *MAGNETIC fields - Abstract
Residual dipolar couplings (RDCs) are increasingly used for high-throughput NMR-based structural studies and to provide long-range angular constraints to validate and refine structures of various molecules determined by X-ray crystallography and NMR spectroscopy. RDCs of a given molecule can be measured in an anisotropic environment that aligns in an external magnetic field. Here, we demonstrate the first application of polymer-based nanodiscs for the measurement of RDCs from nucleic acids. Polymer-based nanodiscs prepared using negatively charged SMA-EA polymer and zwitterionic DMPC lipids were characterized by size-exclusion chromatography, 1H NMR, dynamic light-scattering, and 2H NMR. The magnetically aligned polymer-nanodiscs were used as an alignment medium to measure RDCs from a 13C/15N-labeled fluoride riboswitch aptamer using 2D ARTSY-HSQC NMR experiments. The results showed that the alignment of nanodiscs is stable for nucleic acids and nanodisc-induced RDCs fit well with the previously determined solution structure of the riboswitch. These results demonstrate that SMA-EA-based lipid-nanodiscs can be used as a stable alignment medium for high-resolution structural and dynamical studies of nucleic acids, and they can also be applicable to study various other biomolecules and small molecules in general. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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32. Synaptoneurolipidomics: lipidomics in the study of synaptic function.
- Author
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Ahrends R, Ellis SR, Verhelst SHL, and Kreutz MR
- Abstract
The brain is an exceptionally lipid-rich organ with a very complex lipid composition. Lipids are central in several neuronal processes, including membrane formation and fusion, myelin packing, and lipid-mediated signal transmission. Lipid diversity is associated with the evolution of higher cognitive abilities in primates, is affected by neuronal activity, and is instrumental for synaptic plasticity, illustrating that lipids are not static components of synaptic membranes. Several lines of evidence suggest that the lipid composition of synapses is unique and distinct from other neuronal subcompartments. Here, we delve into the nascent field of synaptoneurolipidomics, offering an overview of current knowledge on the lipid composition of synaptic junctions and technological advances that will allow us to study the impact on synaptic function., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2025
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33. The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.
- Author
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Bruguera ES, Mahoney JP, and Weis WI
- Subjects
- Humans, Protein Binding, Frizzled Receptors metabolism, Frizzled Receptors genetics, Signal Transduction, Animals, Wnt Signaling Pathway, Ligands, Tetraspanins metabolism, Tetraspanins genetics, Eye Proteins metabolism, Eye Proteins genetics, beta Catenin metabolism, beta Catenin genetics, Nerve Tissue Proteins metabolism, Nerve Tissue Proteins genetics
- Abstract
Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling., Competing Interests: EB, JM, WW No competing interests declared, (© 2024, Bruguera et al.)
- Published
- 2025
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34. Charge-Stabilized Nanodiscs as a New Class of Lipid Nanoparticles.
- Author
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Pires IS, Hostetler A, Covarrubias G, Carlo IS, Suggs JR, Kim BJ, Pickering AJ, Gordon E, Irvine DJ, and Hammond PT
- Subjects
- Animals, Mice, Lipids chemistry, Humans, Female, Cell Line, Tumor, Liposomes chemistry, Drug Carriers chemistry, Static Electricity, Micelles, Nanoparticles chemistry
- Abstract
Nanoparticles have the potential to improve disease treatment and diagnosis due to their ability to incorporate drugs, alter pharmacokinetics, and enable tissue targeting. While considerable effort is placed on developing spherical lipid-based nanocarriers, recent evidence suggests that high aspect ratio lipid nanocarriers can exhibit enhanced disease site targeting and altered cellular interactions. However, the assembly of lipid-based nanoparticles into non-spherical morphologies has typically required incorporating additional agents such as synthetic polymers, proteins, lipid-polymer conjugates, or detergents. Here, charged lipid headgroups are used to generate stable discoidal lipid nanoparticles from mixed micelles, which are termed charge-stabilized nanodiscs (CNDs). The ability to generate CNDs in buffers with physiological ionic strength is restricted to lipids with more than one anionic group, whereas monovalent lipids only generate small nanoliposomal assemblies. In mice, the smaller size and anisotropic shape of CNDs promote higher accumulation in subcutaneous tumors than spherical liposomes. Further, the surface chemistry of CNDs can be modified via layer-by-layer (LbL) assembly to improve their tumor-targeting properties over state-of-the-art LbL-liposomes when tested using a metastatic model of ovarian cancer. The application of charge-mediated anisotropy in lipid-based assemblies can aid in the future design of biomaterials and cell-membrane mimetic structures., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)
- Published
- 2024
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35. Observing conformational changes in membrane proteins using cryoEM
- Author
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Pourmal, Sergei
- Subjects
Biophysics ,ABC Transporter ,Cryo-Electron Microscopy ,Membrane Protein ,Multidrug Resistance Protein ,Nanodiscs ,Potassium Channel - Abstract
Proteins are the macromolecular machines responsible for many of life’s fundamental processes. As extended polymers of amino acids, proteins fold into unique three-dimensional shapes in order to carry out their biological roles. Understanding the connection between protein structure and function has been an active area of research for much of the last century, and throughout that time the primary method of structure determination has been X-ray crystallography. Recent advances in the field of cryo-electron microscopy (cryoEM), particularly with respect to electron detectors and algorithms for image processing, have allowed the technique to reach resolutions that rival X-ray diffraction studies. Without the requirement of a crystallized sample, cryoEM opens the possibility of visualizing previously intractable protein targets as well as flexibility and motion within those proteins. My thesis focuses on the application of single particle cryoEM to study membrane proteins in lipidic environments and the conformational changes they undergo. In the first chapter, I present a series of structures of the Bos taurus multidrug resistance protein 4 (MRP4). MRP4 is an ATP binding cassette transporter belonging to the ABCC family and is responsible for the rapid efflux of multiple endogenous and exogenous substrates. Included among these substrates are prostaglandins, a group of biologically active lipid molecules tied to physiological processes as diverse as inflammation and vasoactivity, and whose dysregulation is implicated in pathologies such as cancer and thrombosis. While MRP4 plays a crucial role in localized cell-to-cell signaling as the sole prostaglandin exporter identified in eukaryotes, the molecular details of it’s transport activity are poorly understood. Using single particle cryoEM, I determined five high resolution structures of MRP4 along various steps of the substrate transport cycle. These structures reveal for the first time the basis of MRP4’s affinity for prostaglandins and other organic anions, how substrate binding can stimulate MRP4’s basal ATPase activity several fold, and the conformational changes required for substrate transport. These results broaden our understanding not only of MRP4, but also closely related members of the ABCC family. In the second chapter, I and others describe the novel structure of Arabidopsis thaliana AKT1, a hyperpolarization-activated voltage-gated K+ channel responsible for K+ uptake by the plant’s roots. AKT1 is a Shaker-like channel, and forms homo-tetramers that are under multiple, redundant levels of regulation. Our single particle cryoEM analysis revealed a potential novel form of autoinhibition via a disulfide bond between a soluble N-terminal helix and C-linker. The orientation of this covalent linkage sterically hinders the activation of AKT1’s voltage-sending domain and induces a dramatic restructuring of the tetramer from a C4-symmetric channel into a C2-symmetric one. The transitions between the two conformations of AKT1 found in the C2-symmetric channel resemble those of cyclic-nucleotide gated ion-channels upon cyclic nucleotide binding, suggesting a regulatory role for the previously unreported disulfide bond. Our proposed model of AKT1 autoinhibition provides insights into similar forms of regulation across other hyperpolarization-activated channels.
- Published
- 2023
36. Lipid bilayers determine the allostery but not intrinsic affinity of cAMP binding to pacemaker channels.
- Abstract
A recent preprint abstract discusses the role of lipid bilayers in regulating the binding of cyclic adenosine monophosphate (cAMP) to pacemaker channels in the human heart. The study found that lipid membranes promote cooperative binding of cAMP to HCN channels without changing the intrinsic binding affinity. This research highlights the importance of lipid membranes in enhancing the gating sensitivity of ion channels. The preprint has not yet undergone peer review. [Extracted from the article]
- Published
- 2025
37. Researchers at Northwestern Polytechnic University Report New Data on Protein Assemblies (A Supramolecular Protein Assembly Intrinsically Rescues Memory Deficits In an Alzheimer's Disease Mouse Model).
- Abstract
Researchers at Northwestern Polytechnic University in Shaanxi, China, have developed a new method using supramolecular protein assemblies to address memory deficits in an Alzheimer's disease mouse model. These protein assemblies can cross the blood-brain barrier, recognize and eliminate amyloid plaques, promote neurogenesis, and improve cognitive function. This innovative approach opens up possibilities for designing intelligent supramolecular biomaterials for treating neurodegenerative diseases. The study was supported by the National Natural Science Foundation of China and has been peer-reviewed. [Extracted from the article]
- Published
- 2024
38. Researchers Submit Patent Application, "Surface Plasmon Resonance Sensor Comprising Metal Coated Nanostructures And A Molecularly Imprinted Polymer Layer", for Approval (USPTO 20240402075).
- Subjects
CHEMICAL reagents ,LIQUID chromatography-mass spectrometry ,SURFACE plasmon resonance ,IMPRINTED polymers ,DIELECTRIC materials - Abstract
A patent application has been submitted for a "Surface Plasmon Resonance Sensor" that can detect harmful substances in beverages before consumption. The sensor utilizes nanostructures and a molecularly imprinted polymer layer to create a color change when an analyte of interest is present. This innovative technology aims to provide a proactive and discreet method for detecting harmful agents in liquids. [Extracted from the article]
- Published
- 2024
39. Findings from Massachusetts Institute of Technology Has Provided New Data on Cancer Gene Therapy (Charge-stabilized Nanodiscs As a New Class of Lipid Nanoparticles).
- Subjects
TECHNOLOGICAL innovations ,GENE therapy ,CANCER genes ,CANCER treatment ,REPORTERS & reporting - Abstract
A study conducted by researchers at the Massachusetts Institute of Technology explores the use of charge-stabilized nanodiscs (CNDs) as a new class of lipid nanoparticles for cancer gene therapy. These CNDs, formed from mixed micelles with charged lipid headgroups, show promise in enhancing disease site targeting and altering cellular interactions. The research, funded by various institutions including the National Institutes of Health, highlights the potential of anisotropic lipid-based assemblies in designing biomaterials and cell-membrane mimetic structures for future applications in cancer treatment. [Extracted from the article]
- Published
- 2024
40. Patent Issued for Surface plasmon resonance sensor comprising metal coated nanostructures and a molecularly imprinted polymer layer (USPTO 12140529).
- Subjects
CHEMICAL reagents ,LIQUID chromatography-mass spectrometry ,SURFACE plasmon resonance ,IMPRINTED polymers ,DIELECTRIC materials - Abstract
A patent has been issued for a surface plasmon resonance sensor developed by DrinkSavvy Inc., which aims to detect incapacitating agents in beverages before consumption. The sensor utilizes metal-coated nanostructures and a molecularly imprinted polymer layer to detect analytes of interest in fluid samples, causing a color change upon contact with the analyte. This innovative technology provides a proactive approach to detecting harmful substances in beverages discreetly and efficiently. [Extracted from the article]
- Published
- 2024
41. Researchers from Massachusetts Institute of Technology Provide Details of New Studies and Findings in the Area of Nanodiscs (Magnetoelectric Nanodiscs Enable Wireless Transgene-free Neuromodulation).
- Abstract
Researchers from the Massachusetts Institute of Technology have developed magnetoelectric nanodiscs that enable wireless transgene-free neuromodulation. These nanodiscs, known as MENDs, have shown robust responses to magnetic field stimulation in neurons, allowing for remote control of reward or motor behaviors in genetically intact mice. The study suggests that this minimally invasive neuromodulation technique could have significant applications in neuroscience research and treatment. The research was funded by various organizations including the National Institutes of Health and the European Innovation Council. [Extracted from the article]
- Published
- 2024
42. Studies from Institute for Advanced Research Further Understanding of Nanopharmaceuticals (Vortex Nanodiscs Functionalization to Overcome Macrophage Recognition for Efficient Theragnosis Applications).
- Abstract
The article discusses a study on nanopharmaceuticals conducted by the Institute for Advanced Research, focusing on the functionalization of vortex nanodiscs to overcome macrophage recognition for efficient theragnosis applications. The research highlights the challenges faced by nanotherapeutics in successfully accumulating at target cells due to biological barriers. By synthesizing biocompatible multilayered nanodiscs with inert surface modifications, the study aims to reduce macrophage uptake while maintaining biocompatibility, paving the way for the development of specific nanotherapeutics. The research offers insights into innovative design features that can be incorporated to enhance the effectiveness of nanodiscs in diagnosis and treatment responses. [Extracted from the article]
- Published
- 2024
43. Researchers from Kangwon National University Report on Findings in Cancer (Nanorod/nanodisk-integrated Liquid Crystalline Systems for Starvation, Chemodynamic, and Photothermal Therapy of Cancer).
- Abstract
Researchers from Kangwon National University in Gangwon Do, South Korea, have developed a hybrid gel system combining organic nanorods and inorganic nanodisks for localized cancer therapy. This system integrates starvation therapy, chemodynamic therapy, and photothermal therapy to target cancer cells effectively. The gel, containing Indocyanine green, glucose oxidase, and copper(II) sulfate, showed promising results in suppressing tumor growth and inducing apoptosis in colorectal cancer cells. The study highlights the potential of nanotechnology in improving cancer treatment outcomes. [Extracted from the article]
- Published
- 2024
44. Engineering Substrate-Mediated Localized Surface Plasmons in Gold Nanodiscs
- Author
-
Anisha Chirumamilla, Maria H. Salazar, Deyong Wang, Peter K. Kristensen, Duncan S. Sutherland, Manohar Chirumamilla, Vladimir N. Popok, and Kjeld Pedersen
- Subjects
3D nanostructures ,nanodiscs ,substrate engineering ,localized surface plasmon resonance ,surface enhanced Raman scattering ,Applied optics. Photonics ,TA1501-1820 - Abstract
A variety of nanostructures capable of generating strong local electromagnetic fields (hot spots) in interaction with radiation have been under intensive investigation towards plasmonic applications in surface-enhanced Raman scattering (SERS), biosensing, broadband absorbers, thermophotovoltaics, photocatalysis, etc. In many cases, these nanostructures are formed on a surface or embedded into a near-surface layer of the dielectric substrate, making some part of the field dissipate into the bulk and not contribute to the desired plasmonic functionality. To reduce such losses, the interface between the metallic nanostructures and the dielectric environment should be engineered. In the current work, Au nanodiscs are fabricated on Si posts of very small diameter (pin-shaped structures), enabling them to decouple the strong optical near fields localized at the nanodiscs from the bulk Si substrate. The Si post diameter is optimized by adjusting the gas flow rates in reactive-ion etching, resulting in a minimum post diameter of 20 nm at the nanodisc interface. The effect of this diameter on the localized surface plasmon resonance of the nanodisc is investigated with linear optical spectroscopic measurements, where a significant spectral blue shift of the resonance band is noticed compared to similar discs formed on bulk Si surface. The experimental results are compared with modelling where a 3-fold increase in the electric field enhancement is demonstrated. The fabricated pin-shaped nanostructures are tested in SERS measurements showing a significant increase in the enhancement factor in the order of 106. Thus, this work suggests a way of engineering 3D morphology to tune the substrate influence on the plasmonic properties of nanostructures and to develop efficient nanofabrication technologies.
- Published
- 2023
- Full Text
- View/download PDF
45. Transporter function characterization via continuous-exchange cell-free synthesis and solid supported membrane-based electrophysiology.
- Author
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Dong, Fang, Lojko, Pawel, Bazzone, Andre, Bernhard, Frank, and Borodina, Irina
- Subjects
- *
ELECTROPHYSIOLOGY , *CARRIER proteins , *PROTEIN synthesis , *MEMBRANE proteins , *CELL physiology - Abstract
[Display omitted] • Transporter proteins are essential for cellular function, but their functions are poorly characterized due to the lack of direct assays. • New workflow for transporter functional characterization combines cell-free transporter protein expression and solid supported membrane-based electrophysiology. • The workflow can be executed in five days. • Five transporters from SMR, MFS, Nha, and MC families were functionally expressed and analyzed. • The assay can provide: substrate specificity, kinetic parameters, pH dependency, and mechanistic insights. Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli , and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as K M , I MAX , and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
46. Lipid composition affects the thermal stability of cytochrome P450 3A4 in nanodiscs.
- Author
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Knetsch, Tim G.J. and Ubbink, Marcellus
- Subjects
- *
CYTOCHROME P-450 CYP3A , *SCAFFOLD proteins , *MEMBRANE proteins , *CYTOSKELETAL proteins , *CYTOCHROME P-450 - Abstract
Nanodiscs (NDs), self-assembled lipid bilayers encircled by membrane scaffold proteins (MSPs), offer a versatile platform for the reconstitution of membrane proteins for structural and biochemical investigations. Saturated, isoprenoid lipids are commonly found in thermophiles and have been associated with thermotolerance. To test whether these lipids confer additional stability on ND-incorporated membrane proteins, this study focuses on the thermal stability of human cytochrome P450 3A4 (CYP3A4) inside NDs composed of different phosphocholine lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). NDs were characterized using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and densitometric SDS-PAGE. CYP3A4-DPhPC-NDs were found to comprise three MSP copies instead of the canonical dimer, as reported before for the empty NDs. Rapid, thermally induced unfolding of CYP3A4 inside NDs measured using circular dichroism and differential scanning fluorimetry (nanoDSF) revealed that the CYP3A4 melting temperature was dependent on ND composition. In POPC and DMPC-CYP3A4-NDs the melting temperature was comparable to CYP3A4 without NDs (59 °C). CYP3A4 in DPhPC-NDs showed an increase in melting temperature of 4 °C. Decline in CYP3A4 integrity as well as ND aggregation and disintegration occur at similar rates for all membrane types when subjected to exposure at 37 °C for several hours. The POPC and DMPC- CYP3A4-NDs show significant lipid loss over time, which is not observed for DPhPC-NDs. The results demonstrate that thermally induced denaturation of protein-NDs is a complex, multifaceted process, which is not represented well by rapid thermal unfolding experiments. [Display omitted] • Phytanyl chain Nanodiscs contain three scaffold proteins, offering accessible lipid area for membrane protein reconstitution. • Cytochrome P450 3A4 has a higher melting temperature in phytanyl Nanodiscs than in Nanodiscs containing linear acyl lipids. • Nanodisc thermal stability was measured over time using orthogonal techniques to assess constituent fates and stability. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
47. Advanced applications of Nanodiscs-based platforms for antibodies discovery.
- Author
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Baskakova, Kristina O., Kuzmichev, Pavel K., and Karbyshev, Mikhail S.
- Subjects
- *
CELL receptors , *PROTEIN expression , *MEMBRANE proteins , *DRUG discovery , *PROTEIN engineering - Abstract
Due to their fundamental biological importance, membrane proteins (MPs) are attractive targets for drug discovery, with cell surface receptors, transporters, ion channels, and membrane-bound enzymes being of particular interest. However, due to numerous challenges, these proteins present underutilized opportunities for discovering biotherapeutics. Antibodies hold the promise of exquisite specificity and adaptability, making them the ideal candidates for targeting complex membrane proteins. They can target specific conformations of a particular membrane protein and can be engineered into various formats. Generating specific and effective antibodies targeting these proteins is no easy task due to several factors. The antigen's design, antibody-generation strategies, lead optimization technologies, and antibody modalities can be modified to tackle these challenges. The rational employment of cutting-edge lipid nanoparticle systems for retrieving the membrane antigen has been successfully implemented to simplify the mechanism-based therapeutic antibody discovery approach. Despite the highlighted MP production challenges, this review unequivocally underscores the advantages of targeting complex membrane proteins with antibodies and designing membrane protein antigens. Selected examples of lipid nanoparticle success have been illustrated, emphasizing the potential of therapeutic antibody discovery in this regard. With further research and development, we can overcome these challenges and unlock the full potential of therapeutic antibodies directed to target complex MPs. [Display omitted] • Nanodisc-based platforms emerged as a more advanced approach to antibody discovery and drug development. • Advantages and limitations of current protein expression systems for membrane proteins are described. • Successful implementations of nanodisc-based platforms for antibody discovery against membrane proteins are reviewed. • Prospects of nanodisc-based antibody and vaccine discovery strategies are outlined. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
48. Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
- Author
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Akandé Rouchidane Eyitayo, Axel Boudier-Lemosquet, Stéphane Chaignepain, Muriel Priault, and Stéphen Manon
- Subjects
Bcl-xL ,Bax ,nanodiscs ,cell-free synthesis ,membrane insertion ,MALDI ,Microbiology ,QR1-502 - Abstract
The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.
- Published
- 2023
- Full Text
- View/download PDF
49. Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4.
- Author
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Denisov, Ilia G., Grinkova, Yelena V., McLean, Mark A., Camp, Tyler, and Sligar, Stephen G.
- Subjects
- *
CYTOCHROME P-450 CYP3A , *DRUG interactions , *MIDAZOLAM , *BINDING sites - Abstract
Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
50. Solvophobically‐Driven Merocyanine Dye Assembly: Predominant Dipole‐Dipole Interactions Over Hydrogen‐Bonding.
- Author
-
Rajak, Aritra, Kumar Singh, Ajeet, Roy, Lisa, and Das, Anindita
- Subjects
DIPOLE-dipole interactions ,INTERMOLECULAR forces ,POLAR solvents ,DIPOLE moments ,ELECTROSTATIC interaction ,DIPOLE interactions - Abstract
Merocyanine dye assembly in nonpolar solvents is driven by electrostatic dipole‐dipole interactions, which make hierarchical structures of merocyanine less favorable, due to the compensation of its dipole moment in the discrete antiparallel dimer state. Herein, we describe the self‐assembly of a merocyanine dye (MC−OH) into higher aggregates in aqueous medium (with 10% dioxane) by synergistic effect of dipole‐dipole interactions and strong dispersion forces, which remains underexplored for its known molecular stabilization in polar solvents. Our results reveal that in the crystal packing, strong intermolecular hydrogen (H)‐bonding predominates over the dipole‐dipole interactions, which confines the dye into a head‐to‐head parallel π‐stacked assemblage. When intermolecular H‐bonding in water is curtailed, antiparallel dimers by dipolar interactions become predominant. Several of these antiparallel dimers laterally cluster through solvophobically‐induced π‐stacking and form stable nanodiscs, which exhibit efficient hydrophobic dye sequestering properties. At higher concentration, the nanodiscs are transformed into elongated nanotapes. The computational studies support the experimental findings and emphasize the competing nature of multiple noncovalent interactions in guiding the dye assembly under different conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
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