Conformational trapping of an ABC transporter in polymer lipid nanoparticles.
Conformational trapping of an ABC transporter in polymer lipid nanoparticles.
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DOI：
10.1042/bcj20210312
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发表时间：
2022-01-28
期刊：
影响因子：
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通讯作者：
Dafforn TR
中科院分区：
文献类型：
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作者：
Pollock NL;Lloyd J;Montinaro C;Rai M;Dafforn TR
ATP 结合盒 (ABC) 蛋白作为输入者和输出者在细胞中发挥着重要作用，但作为膜蛋白，它们面临着分离纯且稳定的样品进行研究的众所周知的挑战。解决这个问题的一种方法是使用苯乙烯-马来酸脂质颗粒（SMALP）。苯乙烯-马来酸 (SMA) 可以直接添加到膜上，形成包含膜蛋白和脂质的稳定纳米颗粒。在这里，我们使用 Sav1866（一种充分表征的细菌蛋白）作为一般 ABC 蛋白的代表。我们证明，使用 SMA 可以高产率纯化稳定且单分散的 Sav1866。该蛋白质可用于生物物理表征，表明其整体结构与现有证据一致。然而，与 SMALP 中的其他 ABC 蛋白一样，它不会水解 ATP。 ABC-SMALP 中缺乏 ATP 酶活性可能是由于 SMALP 中蛋白质的构象捕获所致。以受控方式进行的构象捕获是将蛋白质样品稳定为单一构象以进行结构研究的有用工具。由于它们无法水解 ATP，因此纯化后无法使用 ATP 和钒酸盐改变 Sav1866-SMALP 的构象。为了实现 Sav1866–SMALP 的受控捕获，我们证明粗膜中的 Sav1866 可以与 ATP、原钒酸钠和镁一起孵育。随后用 SMA 进行溶解和纯化，生成稳定性增强且处于单一构象状态的 Sav1866-SMALP 样品。该方法可能普遍适用于钒酸盐敏感的ABC蛋白，并克服了SMALP系统对该蛋白家族研究的限制。
ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC–SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866–SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866–SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866–SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.