The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy
The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy
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DOI：
10.1016/j.bbamem.2014.10.018
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发表时间：
2015-02-01
影响因子：
3.4
通讯作者：
Muench, Stephen P.
中科院分区：
文献类型：
--
作者：
Postis, Vincent;Rawson, Shaun;Muench, Stephen P.
尽管最近在解析其结构方面取得了很大进展，但膜蛋白的结构生物学研究仍然面临重大挑战。即使有了新的技术进步，如无规立方相结晶，获得有序的晶体仍然是膜蛋白X射线晶体学研究的一个重大障碍。作为替代，电子显微镜已经显示出能够在中等大小（类似于300 kDa）的膜蛋白中分辨>3.5埃分辨率的细节，而不需要晶体。然而，对于任一方法，常规使用去污剂存在几个问题，包括从其天然膜环境中去除蛋白质对结构的可能影响。作为替代方案，最近已经证明，在不存在去污剂的情况下，使用苯乙烯马来酸共聚物（SMA）可以有效地分离膜蛋白。这种方法产生SMA脂质颗粒（SMALP），其中膜蛋白被聚合物包围的脂质双层的小圆盘包围。在这里，我们使用大肠杆菌二级转运蛋白AcrB作为模型膜蛋白，以证明如何SMALP支架可以用来可视化膜蛋白，嵌入在一个接近天然的脂质环境，通过负染色电子显微镜，产生结构在一个适度的分辨率在短（天）的时间内。此外，我们表明，在SMALP支架内的AcrB比等效的DDM稳定形式显着更活跃。讨论了SMALP支架在电子显微镜下的优势，我们得出的结论是，它们可能被证明是研究膜蛋白结构和功能的重要工具。(C)2014作者由Elsevier B. V.发布。这是CC BY许可下的开放获取文章（http：//creativecommons.org/licenses/by/3.0/）。
Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5 angstrom resolution detail in membrane proteins of modest (similar to 300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).