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A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs

机译：无洗涤剂策略，用于将活性酶复合物从天然生物膜重构为纳米级圆盘

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Background The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored. Results The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes. Conclusions We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.
机译：背景技术将膜蛋白和复合物重构为纳米级脂质双层结构对生物化学和生物物理分析做出了重要贡献。用于进行这种重构的当前方法需要初始的去污剂介导的步骤来溶解和分离膜蛋白。然而，暴露于去污剂会破坏许多膜蛋白的稳定性，并导致功能丧失。两亲共聚物最近已用于在合适的去污剂提取后稳定膜蛋白和复合物。然而，尚未探索这些共聚物直接从天然脂质双层中提取蛋白质用于随后的重构和表征的能力。结果苯乙烯-马来酸（SMA）共聚物可有效溶解分离的线粒体膜和提取的蛋白质复合物。膜复合物与内源性脂质一起重构为聚合物结合的纳米级圆盘。使用呼吸复合物IV作为模型，这些颗粒显示出可以维持多组分电子传输复合物的酶活性。结论我们报告了一种使用SMA共聚物将完全可操作的蛋白质复合物直接从细胞膜重构为纳米级脂质双层的新方法。这种简便的单步操作策略消除了对清洁剂的需求，并产生了适合结构和功能研究的膜复合物。

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