Suitable membrane mimetics are crucial to the performance of 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, which caused 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- to 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 optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (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 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.

中文翻译：

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组装大小可控的磷脂纳米圆盘，用于通过NMR进行膜蛋白的结构研究。

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（当膜支架蛋白围绕脂质双分子层的一小部分时形成）具有类似天然的膜特性。这些已用于各种功能研究，但是通过高分辨率溶液状态NMR光谱对膜的蛋白质进行结构研究，这些膜蛋白通常用于直径为10 nm的纳米圆盘中，但受这些颗粒的高分子量的限制，这导致了较大的分子量NMR线宽。我们最近构建了膜支架蛋白的截短形式，允许制备一系列逐步较小的纳米盘（直径6至8 nm）来克服此限制。这里，我们提出了一个解决方案，用于使用溶液态NMR光谱对膜蛋白进行结构研究的各种尺寸的磷脂纳米盘的组装。我们描述了在纳米光盘中使用大型膜蛋白系统所需的特定同位素标记方案，并提供了有关NMR非均匀采样（NUS）数据采集和高分辨率NMR谱图重建的设置指南。我们讨论了与用于NMR光谱的纳米光盘优化相关的关键点和陷阱，并概述了使用核Overhauser增强光谱（NOESY）光谱，残留偶极耦合（RDC）的高分辨率结构确定和同位素标记的膜蛋白在纳米光盘中的定位策略。 ）和顺磁弛豫增强（PRE）。根据目标蛋白的不同，纳米光盘的组装和纯化可以在12-24小时内完成。尽管此协议的重点是蛋白质NMR，但这些纳米光盘也可用于（低温）电子显微镜（EM）以及小角度X射线和中子散射研究。