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Protocol for Engineered Compositional Asymmetry Within Nanodiscs
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DOI：10.3390/membranes16010044.png)
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摘要
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Membrane proteins remain the most challenging targets for structural characterization, yet their elucidation provides valuable insights into protein function, disease mechanisms, and drug specificity. Structural biology platforms have advanced rapidly in recent years, notably through the development and implementation of nanodiscs—discoidal lipid–protein complexes that encapsulate and solubilize membrane proteins within a controlled, native-like environment. While nanodiscs have become powerful tools for studying membrane proteins, faithfully reconstituting the compositional asymmetry intrinsic to nearly all biological membranes has not yet been achieved. Proper membrane leaflet lipid distribution is critical for accurate protein folding, stability, and insertion. Here, we share a protocol for reconstituting tailored compositional asymmetry within nanodiscs through membrane extraction from giant unilamellar vesicles (GUVs) treated with a leaflet-specific methyl-β-cyclodextrin (mβCD) lipid exchange. Nanodisc asymmetry is verified through a geometric approach: biotin-DPPE-preloaded mβCD engages in lipid exchange with the outer leaflet of POPC GUVs solubilized by the lipid-free membrane scaffold protein (MSP) Δ49ApoA-I to form nanodisc structures. Once isolated, nanodiscs are introduced to the biotin-binding bacterial protein streptavidin. High-speed atomic force microscopy imaging depicts nanodisc–dimer complexes, indicating that biotin-DPPE was successfully reconstituted into a single leaflet of the nanodiscs. This finding outlines the first step toward engineering tailored nanodisc asymmetry and mimicking the native environment of integral proteins—a potentially powerful tool for accurately reconstituting and structurally analyzing integral membrane proteins whose functions are modulated by lipid asymmetry.
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论文数:
5.0K
被引数:
1.6W
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引用论文
The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)天然脂质不对称性对膜插入蛋白 (Perfringolysin O) 构象的影响OAAI
OAAI分享收藏Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape.png)
NATURE CHEMICAL BIOLOGYOAAI
NATURE CHEMICAL BIOLOGYIF13.7
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