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Stephen G Sligar - One of the best experts on this subject based on the ideXlab platform.
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Nanodisc self assembly is thermodynamically reversible and controllable
Soft Matter, 2020Co-Authors: Tyler Camp, Stephen G SligarAbstract:Many highly ordered complex systems form by the spontaneous self-assembly of simpler subunits. An important biophysical tool that relies on self-assembly is the Nanodisc system, which finds extensive use as native-like environments for studying membrane proteins. Nanodiscs are self-assembled from detergent-solubilized mixtures of phospholipids and engineered helical proteins called membrane scaffold proteins (MSPs). Detergent removal results in the formation of nanoscale bilayers stabilized by two MSP "belts." Despite their numerous applications in biology, and contributions from many laboratories world-wide, little is known about the self-assembly process such as when the bilayer forms or when the MSP associates with lipids. We use fluorescence and optical spectroscopy to probe self-assembly at various equilibria defined by the detergent concentration. We show that the bilayer begins forming below the critical micellar concentration of the detergent (10 mM), and the association of MSP and lipids begins at lower detergent levels, showing a dependence on the concentrations of MSP and lipids. Following the dissolution process by adding detergent to purified Nanodiscs demonstrates that the self-assembly is reversible. Our data demonstrate that Nanodisc self-assembly is experimentally accessible, and that controlling the detergent concentration allows exquisite control over the self-assembly reaction. This improved understanding of self-assembly could lead to better functional incorporation of hitherto intractable membrane target proteins.
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microfluidic platform for efficient Nanodisc assembly membrane protein incorporation and purification
Lab on a Chip, 2017Co-Authors: James H Wade, Stephen G Sligar, Joshua D Jones, Ivan L Lenov, Colleen M Riordan, Ryan C BaileyAbstract:The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.
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Nanodiscs in Membrane Biochemistry and Biophysics
Chemical reviews, 2017Co-Authors: Ilia G. Denisov, Stephen G SligarAbstract:Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on the molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system that provides control over size, composition, and specific functional modifications on the nanometer scale. In this review we attempted to combine a comprehensive list of various applications of Nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by Nanodiscs for structural and mechanistic studies of membrane proteins.
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Nanodiscs for structural and functional studies of membrane proteins
Nature Structural & Molecular Biology, 2016Co-Authors: Ilia G. Denisov, Stephen G SligarAbstract:Membrane proteins have long presented a challenge to biochemical and functional studies. In the absence of a bilayer environment, individual proteins and critical macromolecular complexes may be insoluble and may display altered or absent activities. Nanodisc technology provides important advantages for the isolation, purification, structural resolution and functional characterization of membrane proteins. In addition, the ability to precisely control the Nanodisc composition provides a nanoscale membrane surface for investigating molecular recognition events. The use of Nanodiscs is substantially fostering structural and functional studies of membrane protein. This Perspective summarizes the recent use of Nanodiscs as an invaluable tool for the characterization of membrane proteins.
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small angle scattering determination of the shape and localization of human cytochrome p450 embedded in a phospholipid Nanodisc environment
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Nicholas Skargislinge, Ilia G. Denisov, Stephen G Sligar, Ivan L Lenov, Soren Kynde, Lise ArlethAbstract:Membrane proteins reconstituted into phospholipid Nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a Nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast Nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-Nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the Nanodisc and with the large catalytic domain leaning over the outer edge of the Nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.
Ayyalusamy Ramamoorthy - One of the best experts on this subject based on the ideXlab platform.
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Benchmarks of SMA-Copolymer Derivatives and Nanodisc Integrity.
Langmuir : the ACS journal of surfaces and colloids, 2021Co-Authors: Giacomo M. Di Mauro, Carmelo La Rosa, Marcello Condorelli, Ayyalusamy RamamoorthyAbstract:Poly(styrene-co-maleic acid) or SMA and its derivatives, a family of synthetic amphipathic copolymers, are increasingly used to directly solubilize cell membranes to functionally reconstitute membrane proteins in native-like copolymer-lipid Nanodiscs. Although these copolymers act, de facto, like a "macromolecular detergent", the polymer-based lipid-Nanodiscs has been demonstrated to be an excellent membrane mimetic for structural and functional studies of membrane proteins and their complexes by a variety of biophysical and biochemical approaches. In many studies reported in the literature, the choice of the right SMA formulation can depend on a number of factors, and the experimental conditions are typically developed according to a trial-and-error process since each studied system requires adapted protocols. While increasing number of Nanodisc-forming copolymers are reported to be useful and they provide flexibilities in optimizing the sample preparation conditions, it is important to develop a systematic protocol that can be used for various applications. In this context, there is a vital necessity of benchmarking the performances of existing copolymer formulations, assessing crucial parameters for the successful extraction, isolation, and stabilization of membrane proteins. In this study, we compare both copolymers and copolymer-lipid Nanodiscs obtained by SMA-EA with a set of anionic XIRAN copolymer formulations commercially available under the names of SL25010 P, SL30010 P, and SL40005 P. The reported results show how the critical micellar concentration (c.m.c.) of each copolymer is significantly altered in the presence of lipids and confirms the existence of an equilibrium between Nanodisc-bound and "free" or "micellar" copolymer chains in the solution. We believe that these findings can be exploited to optimize studies that involve the necessity of special copolymers, which would not only simplify the applications but also broaden the scope of polymer-based Nanodiscs.
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Metal‐Chelated Polymer Nanodiscs for NMR Studies
Angewandte Chemie (International ed. in English), 2019Co-Authors: Nathaniel Z. Hardin, Thirupathi Ravula, Vojč Kocman, Giacomo M. Di Mauro, Ayyalusamy RamamoorthyAbstract:Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1 ) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene-co-maleic acid) polymer which forms Nanodiscs while showing the ability to chelate metal ions. Cu2+ -chelated Nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipid-Nanodisc components. The chelated Nanodiscs also decrease the proton T1 values for a water-soluble DNA G-quadruplex. These results suggest that polymer Nanodiscs functionalized with paramagnetic tags can be used to speed-up data acquisition from lipid bilayer samples and also to provide structural information from water-soluble biomolecules.
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Self-Assembly of Polymer-Encased Lipid Nanodiscs and Membrane Protein Reconstitution.
The journal of physical chemistry. B, 2019Co-Authors: Bikash R. Sahoo, Takuya Genjo, Kanhu C. Moharana, Ayyalusamy RamamoorthyAbstract:The absence of detergent and curvature makes Nanodiscs excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid Nanodiscs limits their use in the study of the structure, dynamics, and functions of membrane proteins. In this study, we parameterized and optimized the coarse-graining (CG) bead mapping for two differently charged and functionalized copolymers, containing styrene–maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed Nanodisc formation (
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Self-Assembly of Polymer-Encased Lipid Nanodiscs and Membrane Protein Reconstitution
2019Co-Authors: Bikash R. Sahoo, Takuya Genjo, Kanhu C. Moharana, Ayyalusamy RamamoorthyAbstract:The absence of detergent and curvature makes Nanodiscs excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid Nanodiscs limits their use in the study of the structure, dynamics, and functions of membrane proteins. In this study, we parameterized and optimized the coarse-graining (CG) bead mapping for two differently charged and functionalized copolymers, containing styrene–maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed Nanodisc formation (
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Alzheimer's amyloid-beta intermediates generated using polymer-Nanodiscs.
Chemical communications (Cambridge England), 2018Co-Authors: Bikash R. Sahoo, Sarah J. Cox, Takuya Genjo, Michael E. Bekier, Andrea K. Stoddard, Magdalena I. Ivanova, Kazuma Yasuhara, Carol A. Fierke, Yanzhuang Wang, Ayyalusamy RamamoorthyAbstract:Polymethacrylate-copolymer (PMA) encased lipid-Nanodiscs (∼10 nm) and macro-Nanodiscs (>15 nm) are used to study Aβ1–40 aggregation. We demonstrate that PMA-Nanodiscs form a ternary association with Aβ and regulate its aggregation kinetics by trapping intermediates. Results demonstrating the reduced neurotoxicity of Nanodisc-bound Aβ oligomers are also reported.
Ilia G. Denisov - One of the best experts on this subject based on the ideXlab platform.
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Nanodiscs in Membrane Biochemistry and Biophysics
Chemical reviews, 2017Co-Authors: Ilia G. Denisov, Stephen G SligarAbstract:Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on the molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system that provides control over size, composition, and specific functional modifications on the nanometer scale. In this review we attempted to combine a comprehensive list of various applications of Nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by Nanodiscs for structural and mechanistic studies of membrane proteins.
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Nanodiscs for structural and functional studies of membrane proteins
Nature Structural & Molecular Biology, 2016Co-Authors: Ilia G. Denisov, Stephen G SligarAbstract:Membrane proteins have long presented a challenge to biochemical and functional studies. In the absence of a bilayer environment, individual proteins and critical macromolecular complexes may be insoluble and may display altered or absent activities. Nanodisc technology provides important advantages for the isolation, purification, structural resolution and functional characterization of membrane proteins. In addition, the ability to precisely control the Nanodisc composition provides a nanoscale membrane surface for investigating molecular recognition events. The use of Nanodiscs is substantially fostering structural and functional studies of membrane protein. This Perspective summarizes the recent use of Nanodiscs as an invaluable tool for the characterization of membrane proteins.
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small angle scattering determination of the shape and localization of human cytochrome p450 embedded in a phospholipid Nanodisc environment
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Nicholas Skargislinge, Ilia G. Denisov, Stephen G Sligar, Ivan L Lenov, Soren Kynde, Lise ArlethAbstract:Membrane proteins reconstituted into phospholipid Nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a Nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast Nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-Nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the Nanodisc and with the large catalytic domain leaning over the outer edge of the Nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.
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Homotropic cooperativity of monomeric cytochrome P450 3A4
Archives of Biochemistry and Biophysics, 2010Co-Authors: Bradley J. Baas, Ilia G. Denisov, Stephen G SligarAbstract:Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidatesmore » new properties of the monomeric form.« less
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Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer Nanodiscs.
Methods in enzymology, 2009Co-Authors: Tasha K. Ritchie, Ilia G. Denisov, Timothy H Bayburt, Yelena V. Grinkova, Joseph K. Zolnerciks, William M. Atkins, Stephen G SligarAbstract:Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodopsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together, these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.
Michael T. Marty - One of the best experts on this subject based on the ideXlab platform.
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Chemical Additives Enable Native Mass Spectrometry Measurement of Membrane Protein Oligomeric State within Intact Nanodiscs
2018Co-Authors: James E. Keener, Deseree J. Reid, Dane Evan Zambrano, Guozhi Zhang, Ciara K. Zak, Bhushan S. Deodhar, Jeanne E. Pemberton, James S. Prell, Michael T. MartyAbstract:Membrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein Nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but previous native MS of membrane proteins in Nanodiscs has been limited by the intermediate stability of Nanodiscs. It is difficult to eject membrane proteins from Nanodiscs for native MS but also difficult to retain intact Nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein Nanodisc complexallowing measurement of the membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of Nanodiscs under different ESI conditions opens new applications for native MS of Nanodiscs
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Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry
Analytical chemistry, 2017Co-Authors: Deseree J. Reid, James E. Keener, Andrew P. Wheeler, Dane Evan Zambrano, Jessica M. Diesing, Maria Reinhardt-szyba, Alexander Makarov, Michael T. MartyAbstract:Lipoprotein Nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of Nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of Nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect Nanodisc formation but shift the masses of Nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt Nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.
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Interpretation and Deconvolution of Nanodisc Native Mass Spectra
Journal of the American Society for Mass Spectrometry, 2013Co-Authors: Michael T. Marty, Hao Zhang, Weidong Cui, Michael L. Gross, Stephen G SligarAbstract:Nanodiscs are a promising system for studying gas-phase and solution complexes of membrane proteins and lipids. We previously demonstrated that native electrospray ionization allows mass spectral analysis of intact Nanodisc complexes at single lipid resolution. This report details an improved theoretical framework for interpreting and deconvoluting native mass spectra of Nanodisc lipoprotein complexes. In addition to the intrinsic lipid count and charge distributions, Nanodisc mass spectra are significantly shaped by constructive overlap of adjacent charge states at integer multiples of the lipid mass. We describe the mathematical basis for this effect and develop a probability-based algorithm to deconvolute the underlying mass and charge distributions. The probability-based deconvolution algorithm is applied to a series of dimyristoylphosphatidylcholine Nanodisc native mass spectra and used to provide a quantitative picture of the lipid loss in gas-phase fragmentation.
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Nanodisc solubilized membrane protein library reflects the membrane proteome
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Michael T. Marty, Kyle C Wilcox, William L Klein, Stephen G SligarAbstract:The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.
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Native mass spectrometry characterization of intact Nanodisc lipoprotein complexes.
Analytical chemistry, 2012Co-Authors: Michael T. Marty, Hao Zhang, Weidong Cui, Robert E. Blankenship, Michael L. Gross, Stephen G SligarAbstract:We describe here the analysis of Nanodisc complexes by using native mass spectrometry (MS) to characterize their molecular weight (MW) and polydispersity. Nanodiscs are nanoscale lipid bilayers that offer a platform for solubilizing membrane proteins. Unlike detergent micelles, Nanodiscs are native-like lipid bilayers that are well-defined and potentially monodisperse. Their mass spectra allow peak assignment based on differences in the mass of a single lipid per complex. Resultant masses agree closely with predicted values and demonstrate conclusively the narrow dispersity of lipid molecules in the Nanodisc. Fragmentation with collisionally activated dissociation (CAD) or electron-capture dissociation (ECD) shows loss of a small number of lipids and eventual collapse of the Nanodisc with release of the scaffold protein. These results provide a foundation for future studies utilizing Nanodiscs as a platform for launching membrane proteins into the gas phase.
Lise Arleth - One of the best experts on this subject based on the ideXlab platform.
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structure and dynamics of a Nanodisc by integrating nmr saxs and sans experiments with molecular dynamics simulations
eLife, 2020Co-Authors: Tone Bengtsen, Søren Roi Midtgaard, Nicolai Tidemand Johansen, Viktor L Holm, Lisbeth Ravnkilde Kjolbye, Giulio Tesei, Sandro Bottaro, Birgit Schiott, Lise ArlethAbstract:Nanodiscs are membrane mimetics that consist of a protein belt surrounding a lipid bilayer, and are broadly used for characterization of membrane proteins. Here, we investigate the structure, dynamics and biophysical properties of two small Nanodiscs, MSP1D1ΔH5 and ΔH4H5. We combine our SAXS and SANS experiments with molecular dynamics simulations and previously obtained NMR and EPR data to derive and validate a conformational ensemble that represents the structure and dynamics of the Nanodisc. We find that it displays conformational heterogeneity with various elliptical shapes, and with substantial differences in lipid ordering in the centre and rim of the discs. Together, our results reconcile previous apparently conflicting observations about the shape of Nanodiscs, and pave the way for future integrative studies of larger complex systems such as membrane proteins embedded in Nanodiscs.
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structure and dynamics of a lipid Nanodisc by integrating nmr saxs and sans experiments with molecular dynamics simulations
bioRxiv, 2020Co-Authors: Tone Bengtsen, Søren Roi Midtgaard, Nicolai Tidemand Johansen, Viktor L Holm, Lisbeth Ravnkilde Kjolbye, Giulio Tesei, Sandro Bottaro, Birgit Schiott, Lise ArlethAbstract:Nanodiscs are membrane mimetics that consist of a protein belt surrounding a lipid bilayer, and are broadly used for characterization of membrane proteins. Here, we investigate the structure, dynamics and biophysical properties of two small Nanodiscs, MSP1D1ΔH5 and ΔH4H5. We combine our SAXS and SANS experiments with molecular dynamics simulations and previously obtained NMR and EPR data to derive and validate a conformational ensemble that represents the structure and dynamics of the Nanodisc. We find that it displays conformational heterogeneity with various elliptical shapes, and with substantial differences in lipid ordering in the centre and rim of the discs. Together, our results reconcile previous apparently conflicting observations about the shape of Nanodiscs, and paves the way for future integrative studies of larger complex systems such as membrane proteins embedded in Nanodiscs.
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Comprehensive Study of the Self-Assembly of Phospholipid Nanodiscs: What Determines Their Shape and Stoichiometry?
Langmuir : the ACS journal of surfaces and colloids, 2018Co-Authors: Nicholas Skar-gislinge, Nicolai Tidemand Johansen, Rasmus Høiberg-nielsen, Lise ArlethAbstract:Phospholipid Nanodiscs have quickly become a widely used platform for studies of membrane proteins. However, the molecular self-assembly process that ultimately should place a membrane protein inside a Nanodisc is not well understood. This poses a challenge for a successful high-yield reconstitution of general membrane proteins into Nanodiscs. In the present work, the self-assembly process of POPC-MSP1D1 Nanodiscs was carefully investigated by systematically modulating the reconstitution parameters and probing the effect with a small-angle X-ray scattering analysis of the resulting Nanodiscs. First, it was established that Nanodiscs prepared using the standard protocol followed a narrow but significant size distribution and that the formed Nanodiscs were stable at room temperature over a time range of about a week. Systematic variation of the POPC/MSP1D1 stoichiometry of the reconstitution mixture showed that a ratio of less than 75:1 resulted in lipid-poor Nanodiscs, whereas ratios of 75:1 and larger res...
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small angle scattering determination of the shape and localization of human cytochrome p450 embedded in a phospholipid Nanodisc environment
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Nicholas Skargislinge, Ilia G. Denisov, Stephen G Sligar, Ivan L Lenov, Soren Kynde, Lise ArlethAbstract:Membrane proteins reconstituted into phospholipid Nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a Nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast Nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-Nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the Nanodisc and with the large catalytic domain leaning over the outer edge of the Nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.
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Lipid-Protein Interactions in Nanodiscs: How to Enhance Stability
Biophysical Journal, 2012Co-Authors: Maria Wadsäter, Marité Cárdenas, Selma Maric, Kell Mortensen, Lise Arleth, Stine Rønholt, Nicholas Skar-gislinge, Søren Roi Midtgaard, Robert O. Ryan, Jens B. SimonsenAbstract:Lipid-protein interactions can function as “co-factors” that affect the properties / function of transmembrane proteins. Herein, the interaction between anionic dimyristoylphosphatidylglycerol (DMPG) and zwitterionic dimyristoylphosphatidylcholine (DMPC) with the amphiphatic membrane scaffold protein (MSP), were studied. Two 25 kDa MSP wrap around the circumference of discoidal bilayer in a belt-like manner to form a Nanodisc [1,2]. The membrane-like structure of Nanodiscs has been used for reconstitution of membrane proteins in a native-like environment. Differential scanning calorimetry was employed to characterize lipid-protein interactions in these particles by evaluating changes in MSP denaturation temperature and lipid gel-liquid phase transition as a function of Nanodisc lipid composition and ionic strength. Small-angle X-ray scattering and size-exclusion chromatography were used to determine the overall structure of the Nanodisc. We suggest the Nanodisc lipid is divided into a lipid rim that interacts with the internal face of the MSP helical segments, while the centrally located Nanodisc lipids maintain a more bulk-like lipid behavior. This finding is important for reconstitution of membrane proteins since the presence of a ‘lipid rim’ serves to prevent contact between the membrane protein and the MSP. Furthermore, the presence of two distinct lipid environments reduces the available area for reconstituted membrane proteins in the Nanodisc. We also show that the negatively charged DMPG has a higher preference for the rim due to its negatively charged headgroup. Finally, we conclude that DMPG stabilizes the Nanodisc in a twofold manner: i) DMPG ’freezes’ the MSP conformation preventing flexibility / dissociation that may lead to aggregation. ii) DMPG also contributes to prevention of aggregation due to electrostatic repulsion between the negatively charged lipids on neighboring Nanodiscs.[1] T.H. Bayburt et al.: Nano Letters 2 (2002) 853.[2] N. Skar-Gislinge and J.B. Simonsen et al.: JACS 132 (2010) 13713.
