Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.
Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.
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DOI：
10.1038/nprot.2017.094
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发表时间：
2018-01
期刊：
影响因子：
14.8
通讯作者：
Wagner G
中科院分区：
文献类型：
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作者：
Hagn F;Nasr ML;Wagner G
合适的膜模拟物对于进行膜蛋白的结构和功能研究至关重要。磷脂纳米盘（当膜支架蛋白包围脂质双层的一小部分时形成）具有天然样膜性质。这些已被用于各种功能的研究，但结构的研究，通过高分辨率的溶液状态NMR光谱的膜蛋白在常用的纳米盘直径为10 nm的限制，这些颗粒的高分子量造成不利的大NMR线宽。我们最近构建了截短版本的膜支架蛋白，允许制备一系列逐步较小的纳米盘（直径6-8 nm）以克服这一限制。在这里，我们提出了一个协议的组装磷脂纳米盘的各种大小的膜蛋白的结构研究与溶液状态的NMR光谱。我们描述了特定的同位素标记方案，需要与大膜蛋白质系统在nanodisks工作，并提供指导方针的NMR非均匀采样（NUS）数据采集和高分辨率NMR光谱重建的设置。我们讨论的关键点和陷阱有关优化nanodisces的NMR光谱和概述的高分辨率的结构测定和定位的同位素标记的膜蛋白在nanodisces使用NOESY光谱，残留偶极耦合（RDC）和顺磁弛豫增强（PREs）的战略。根据目标蛋白的不同，纳米盘的组装和纯化可以在12 - 24小时内完成。虽然该协议的重点是蛋白质NMR，但这些纳米盘也可用于（冷冻）EM和小角度X射线和中子散射研究。
Suitable membrane mimetics are crucial for performing structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly-used nanodiscs of 10 nm diameter were limited by the high molecular weight of these particles causing unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein allowing the preparation of a range of stepwise smaller nanodiscs (6-8 nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope labeling schemes required for working with large membrane protein systems in nanodiscs and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimizing nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using NOESY spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12 – 24h. While the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) EM and small-angle X-ray and neutron scattering studies.
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DOI：
10.1073/pnas.1604125113
发表时间：
2016-06-28
影响因子：
11.1
作者：
Goricanec, David;Stehle, Ralf;Hagn, Franz
通讯作者：
Hagn, Franz
影响因子：
4.8
作者：
Eggensperger, Sabine;Fisette, Olivier;Tampe, Robert
通讯作者：
Tampe, Robert
影响因子：
2.7
作者：
Ayala, Isabel;Sounier, Remy;Boisbouvier, Jerome
通讯作者：
Boisbouvier, Jerome
影响因子：
5.7
作者：
Etzkorn, Manuel;Raschle, Thomas;Wagner, Gerhard
通讯作者：
Wagner, Gerhard
影响因子：
14.8
作者：
通讯作者：
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