A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs

Danielczak, Bartholomäus; Rasche, Marie; Lenz, Julia; Patallo, Eugenio P.; Weyrauch, Sophie; Mahler, Florian; Agbadaola, Michael T.; Meister, Annette; Babalola, Jonathan O.; Vargas, Carolyn; Kolář, Čenêǩ; Keller, Sandro

doi:10.1039/d1nr03811g

Cited by 19 publications

(46 citation statements)

References 54 publications

(170 reference statements)

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“…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%

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: Introductionmentioning

confidence: 99%

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

et al. 2022

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“…[21][22][23][24][25][26][27][28] Apart from SMA and SMA-like copolymers, several other types of amphiphilic copolymers for membrane protein isolation were developed. [29] These included diisobutylene/maleic acid copolymers (DIBMA) and their derivatives, [16,[30][31][32][33] alkylamine-modified poly(acrylic acid) (APAA), [34] methacroylcholine chloride/butyl methacrylate copolymers (PMA), [35] acrylic acid/styrene copolymers (AASTY), [14,36] modified inulin, [37,38] methylstilbene/maleic acid copolymers (STMA), [39] and cycloalkylamine-modified poly(acrylic acid) (CyclAPol). [40] In addition, in a very recent comprehensive study, Kopf et al presented a number of SMA derivatives with diverse substitution mainly at the aromatic ring, as well as acrylic acid copolymers with substituted styrenes.…”

Section: Introductionmentioning

confidence: 99%

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight

Janata

Čadová

Angelisová

et al. 2022

Macromolecular Bioscience

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Low‐molecular weight (MW) amphiphilic copolymers have been recently introduced as a powerful tool for the detergent‐free isolation of cell membrane proteins. Herein, a screening approach is used to identify a new copolymer type for this application. Via a two‐step ATRP/acidolysis procedure, a 3 × 3 matrix of well‐defined poly[(butyl methacrylate)‐co‐(methacrylic acid)] copolymers (denoted BMAA) differing in their MW and ratio of hydrophobic (BMA) and hydrophilic (MAA) units is prepared. Subsequently, using the biologically relevant model (T‐cell line Jurkat), two compositions of BMAA copolymers are identified that solubilize cell membranes to an extent comparable to the industry standard, styrene‐maleic acid copolymer (SMA), while avoiding the potentially problematic phenyl groups. Surprisingly, while only the lowest‐MW variant of the BMA/MAA 2:1 composition is effective, all the copolymers of the BMA/MAA 1:1 composition are found to solubilize the model membranes, including the high‐MW variant (MW of 14 000). Importantly, the density gradient ultracentrifugation/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis/Western blotting experiments reveal that the BMA/MAA 1:1 copolymers disintegrate the Jurkat membranes differently than SMA, as demonstrated by the different distribution patterns of two tested membrane protein markers. This makes the BMAA copolymers a useful tool for studies on membrane microdomains differing in their composition and resistance to membrane‐disintegrating polymers.

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“…Thus, the first step before purifying any membrane protein is to know the net charge of the target protein at a given pH of a buffer being used for purification/reconstitution and select the polymer accordingly. The recently developed anionic glycol-DIBMA with a decreased charge density showed better membrane solubilization than highly-charged DIBMA [ 107 ] ( Figure 13 ). Thus, the inefficient membrane solubilization by SMA-based polymers ( Figure 14 ) was because of charge-charge repulsions with anionic lipids in a membrane.…”

Section: Limitations Of Ionic Polymersmentioning

confidence: 99%

“…While research in this area continues to develop novel nanodisc-forming molecules (such as amphipathic polymers and peptides), these already reported polymers (including cationic, anionic, zwitterionic, and non-ionic) render studies on most (if not all) membrane proteins. An additional list of reported polymers can be found in the literature ([ 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 ]) and on the SMALP website ( , accessed on July 25 2022). The polymer structures were generated using ChemDraw [19.1.1.21].…”

Section: Figurementioning

confidence: 99%

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Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment

Krishnarjuna

Ramamoorthy

2022

Biomolecules

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Atomic-resolution structural studies of membrane-associated proteins and peptides in a membrane environment are important to fully understand their biological function and the roles played by them in the pathology of many diseases. However, the complexity of the cell membrane has severely limited the application of commonly used biophysical and biochemical techniques. Recent advancements in NMR spectroscopy and cryoEM approaches and the development of novel membrane mimetics have overcome some of the major challenges in this area. For example, the development of a variety of lipid-nanodiscs has enabled stable reconstitution and structural and functional studies of membrane proteins. In particular, the ability of synthetic amphipathic polymers to isolate membrane proteins directly from the cell membrane, along with the associated membrane components such as lipids, without the use of a detergent, has opened new avenues to study the structure and function of membrane proteins using a variety of biophysical and biological approaches. This review article is focused on covering the various polymers and approaches developed and their applications for the functional reconstitution and structural investigation of membrane proteins. The unique advantages and limitations of the use of synthetic polymers are also discussed.

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A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs

Abstract: Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among...

Cited by 19 publications

References 54 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

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight

Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment

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