Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins.
Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins.
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DOI：
10.1021/ja310901f
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发表时间：
2013-02-06
影响因子：
15
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Hagn, Franz;Etzkorn, Manuel;Raschle, Thomas;Wagner, Gerhard
膜蛋白的结构研究仍然受到寻找合适的膜模拟介质以维持蛋白质结构和功能的困难。磷脂纳米盘似乎有望克服含有洗涤剂环境的固有问题。虽然纳米盘可以提供接近自然的环境，但大颗粒使其在用溶液核磁共振进行膜蛋白结构分析中的常规使用变得复杂。在这里，我们介绍了由较短的ApoA-I蛋白变体组装而成的纳米盘，这些变异体的直径明显较小，并表明所得到的盘为通过核磁共振确定膜蛋白的结构提供了关键的改进。以细菌外膜蛋白OmpX为例，我们证明了小的纳米盘尺寸、高的蛋白质和脂的氚水平以及先进的非均匀核磁共振采样方法的结合使得在无洗涤剂的、近天然的脂双层环境中能够进行多孔膜蛋白的核磁共振指定以及高分辨率的结构确定。通过将这种方法应用于细菌视紫红质，我们发现我们的更小的纳米盘也可以用于重要的七跨膜螺旋蛋白的结构表征。我们的一套随后直径较小的工程纳米盘可用于筛选中小型膜蛋白的最佳核磁共振谱质量，同时仍可提供功能环境。除了它们在从头结构测定方面的关键改进之外，由于它们的尺寸较小，这些纳米盘能够研究膜蛋白与其(可溶)伙伴蛋白之间的相互作用，而不受可能扰乱生物相关相互作用的洗涤剂的存在的影响。
Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent containing environments. While nanodiscs can offer a near native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin we show that our smaller nanodiscs can also be beneficial for the structural characterization of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for small to medium sized membrane proteins while still providing a functional environment. In addition to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of interactions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biological relevant interactions.
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影响因子：
15
作者：
Hyberts, Sven G.;Takeuchi, Koh;Wagner, Gerhard
通讯作者：
Wagner, Gerhard
DOI：
10.1073/pnas.94.23.12291
发表时间：
1997-11-11
影响因子：
11.1
作者：
Borhani, DW;Rogers, DP;Brouillette, CG
通讯作者：
Brouillette, CG
DOI：
10.1073/pnas.051629298
发表时间：
2001-02-27
影响因子：
11.1
作者：
Fernández, C;Adeishvili, K;Wüthrich, K
通讯作者：
Wüthrich, K
影响因子：
8.6
作者：
Hyberts, Sven G.;Arthanari, Haribabu;Wagner, Gerhard
通讯作者：
Wagner, Gerhard
DOI：
10.1007/s00249-010-0626-7
发表时间：
2011-01-01
影响因子：
2
作者：
Choutko, Alexandra;Glaettli, Alice;van Gunsteren, Wilfred F.
通讯作者：
van Gunsteren, Wilfred F.