Electroneutral Polymer Nanodiscs Enable Interference‐Free Probing of Membrane Proteins in a Lipid‐Bilayer Environment

Glueck, David; Grethen, Anne; Das, Manabendra; Mmeka, Ogochukwu Patricia; Patallo, Eugenio P.; Meister, Annette; Rajender, Ritu; Kins, Stefan; Räschle, Markus; Victor, Julian; Chu, Constance R.; Etzkorn, Manuel; Köck, Zoe; Bernhard, Frank; Babalola, Jonathan O.; Vargas, Carolyn; Keller, Sandro

doi:10.1002/smll.202202492

Cited by 20 publications

(38 citation statements)

References 70 publications

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“…Natural CL contains a mixture of disaturated, diunsaturated, and saturated/unsaturated acyl chains (Figure S1). Sulfo-DIBMA was synthesized as described before . Methanol (MeOH) (99.8%), HPLC-grade chloroform (CHCl 3 ), 1,4-dithiothreitol (DTT) (99%, p.a.…”

Section: Methodsmentioning

confidence: 99%

“…Sulfo-DIBMA was synthesized as described before. 16 Methanol (MeOH) (99.8%), HPLC-grade chloroform (CHCl 3 ), 1,4-dithiothreitol (DTT) (99%, p.a. ), D-sucrose (99.5%), dextrin, dipotassium hydrogen phosphate trihydrate (K 2 HPO 4 •3H 2 O), ethylenediaminetetraacetic acid (EDTA) (99%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (99.5%), iron(III) sulfate hydrate (80%), magnesium chloride hexahydrate (MgCl 2 •H 2 O) (99%), magnesium sulfate heptahydrate (MgSO 4 •7H 2 O) (99%), peptone, sodium chloride (NaCl) (99.5%), potassium chloride (KCl) (99.5%), sodium dodecyl sulfate (SDS), yeast extract, acetic acid (99%), and tris-(hydroxymethyl)aminomethane (Tris) were purchased from Carl Roth (Karlsruhe, Germany).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Polymers such as styrene/maleic acid (SMA) or diisobutylene/maleic acid (DIBMA) are able to extract membrane proteins along with a small patch of their native lipid environment, encapsulating them in lipid-bilayer nanodiscs. These nanodiscs allow for the identification and investigation of membrane proteins in a native-like environment, possibly retaining bound ligands, protein–protein, and protein–lipid interactions, thus allowing a closer-to-native view onto membrane proteins. , Recently, the toolbox of amphiphilic polymers has been expanded, resulting in various new polymers designed to overcome earlier limitations such as low pH stability, tolerance toward divalent metal ions, or poor solubilization efficiency. − Because of its electroneutrality and good solubilization efficiency toward complex lipid compositions, the zwitterionic polymer Sulfo-DIBMA has great potential to be applied to complex membrane environments such as those isolated directly from living cells …”

Section: Introductionmentioning

confidence: 99%

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Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

et al. 2022

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New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein−protein and protein−lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous�as opposed to overexpressed�membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane−protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane−protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane−protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

et al. 2022

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“…Similarly, when less common solubilisation conditions are required, such as low pH or high salt, our studies identify PMA as the optimal polymer to start with, alongside (beyond the scope of this paper) the modified SMA variants SMA-QA or SMI which have been shown to possess a similar insensitivity to low pH and high salt concentrations (Hall et al 2018 ; Ravula et al 2018 ). The further development polymers such as SMA-QA (Ravula et al 2018 ), Sulfo-SMA/Sulfo-DIBMA (Glueck et al 2022 ), and Glyco-DIBMA (Danielczak et al 2022 ) highlights the requirement for standardised characterizations, such as those presented in this work.…”

Section: Discussionmentioning

confidence: 98%

A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes

2023

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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.

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“…Glyco-DIBMA retains solubility similar to that of DIBMA, a similar UV absorption spectrum, and a smaller amount of negative charge. Indeed, the electrostatic repulsion between the polymer chains has been suggested to induce the broad size distribution and larger size of DIBMALPs compared to those of SMALPs, Glyco-DIBMALPs, and other neutral polymers. ,− As recently reported, Glyco-DIBMALPs are stable at physiological pH and ionic strength and in the presence of divalent cations. The formation of Glyco-DIBMALPs with different synthetic phospholipids was tested together with the application of this co-polymer for the direct extraction of membrane proteins from Escherichia coli cells …”

Section: Introductionmentioning

confidence: 92%

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles

et al. 2023

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Different amphiphilic co-polymers have been introduced to produce polymer–lipid particles with nanodisc structure composed of an inner lipid bilayer and polymer chains self-assembled as an outer belt. These particles can be used to stabilize membrane proteins in solution and enable their characterization by means of biophysical methods, including small-angle X-ray scattering (SAXS). Some of these co-polymers have also been used to directly extract membrane proteins together with their associated lipids from native membranes. Styrene/maleic acid and diisobutylene/maleic acid are among the most commonly used co-polymers for producing polymer–lipid particles, named SMALPs and DIBMALPs, respectively. Recently, a new co-polymer, named Glyco-DIBMA, was produced by partial amidation of DIBMA with the amino sugar N-methyl-d-glucosamine. Polymer–lipid particles produced with Glyco-DIBMA, named Glyco-DIBMALPs, exhibit improved structural properties and stability compared to those of SMALPs and DIBMALPs while retaining the capability of directly extracting membrane proteins from native membranes. Here, we characterize the structure and lipid composition of Glyco-DIBMALPs produced with either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Glyco-DIBMALPs were also prepared with mixtures of either POPC or DMPC and cholesterol at different mole fractions. We estimated the lipid content in the Glyco-DIBMALPs and determined the particle structure and morphology by SAXS. We show that the Glyco-DIBMALPs are nanodisc-like particles whose size and shape depend on the polymer/lipid ratio. This is relevant for designing nanodisc particles with a tunable diameter according to the size of the membrane protein to be incorporated. We also report that the addition of >20 mol % cholesterol strongly perturbed the formation of Glyco-DIBMALPs. Altogether, we describe a detailed characterization of the Glyco-DIBMALPs, which provides relevant inputs for future application of these particles in the biophysical investigation of membrane proteins.

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Electroneutral Polymer Nanodiscs Enable Interference‐Free Probing of Membrane Proteins in a Lipid‐Bilayer Environment

Cited by 20 publications

References 70 publications

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles

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