Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples.
Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples.
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DOI：
10.1038/nprot.2013.129
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发表时间：
2013-11
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
固体核磁共振光谱已经成功地用于表征膜蛋白的结构和动力学，以及它们与脂质双层中其他蛋白质的相互作用。这样的环境通常是实现原生结构所必需的。样品制备是成功的关键。在这里，我们提出了一个强大的协议，导致高质量的膜蛋白样品魔角旋转和定向样品固态核磁共振的详细描述。该过程使用两种蛋白进行演示：CrgA（两个跨膜螺旋）和rv1861（三个跨膜螺旋），均来自结核分枝杆菌。手术的成功取决于两点。首先，对于两种核磁共振实验的样品，蛋白质从洗涤剂环境重构到将其纳入脂质体的环境导致“完全”去除洗涤剂。其次，对于定向样品，适当的脱水，然后再水合蛋白脂质体是必不可少的。采用该方案，可在10 d内获得魔角旋转核磁共振的蛋白脂质体样品和取向样品核磁共振的均匀排列样品（取向嵌合度<1°）。
Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. such an environment is often necessary for achieving native-like structures. sample preparation is the key to this success. Here we present a detailed description of a robust protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. the procedure is demonstrated using two proteins: CrgA (two transmembrane helices) and rv1861 (three transmembrane helices), both from Mycobacterium tuberculosis. the success of this procedure relies on two points. First, for samples for both types of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in ‘complete’ removal of detergent. second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d.
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影响因子：
5.6
作者：
Bhate MP;Wylie BJ;Tian L;McDermott AE
通讯作者：
McDermott AE
影响因子：
8
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Korepanova, A;Gao, FP;Cross, TA
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Cross, TA
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ASLANIDIS, C;DEJONG, PJ
通讯作者：
DEJONG, PJ
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56.9
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通讯作者：
CROSS, TA
影响因子：
1.6
作者：
Korepanova, A.;Moore, J. D.;Gao, F.
通讯作者：
Gao, F.