Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, United States.
J Am Chem Soc. 2013 Feb 6;135(5):1919-25. doi: 10.1021/ja310901f. Epub 2013 Jan 25.
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 biologically relevant interactions.
膜蛋白的结构研究仍然受到寻找合适的模拟膜环境的困难的阻碍，这种环境可以维持蛋白质的结构和功能。磷脂纳米盘似乎有希望克服含有去污剂的环境所固有的问题。虽然纳米盘可以提供接近天然的环境，但较大的颗粒大小使其在膜蛋白的结构分析中常规使用变得复杂，特别是在溶液 NMR 方面。在这里，我们介绍了由较短的载脂蛋白 A-I 蛋白变体组装而成的纳米盘，其直径明显较小，并表明所得的盘对于通过 NMR 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，我们证明了小纳米盘尺寸、蛋白质和脂质的高氘化水平以及先进的非均匀 NMR 采样方法的结合，能够实现 NMR 共振分配以及在无去污剂、接近天然的脂质双层环境中对多跨膜蛋白进行高分辨率结构测定。通过将这种方法应用于菌紫质，我们表明我们较小的纳米盘对于重要的七跨膜螺旋蛋白类的结构特征化也可能是有益的。我们的一系列随后直径更小的工程化纳米盘可用于筛选小至中等大小的膜蛋白的最佳 NMR 光谱质量，同时仍提供功能性环境。除了对从头确定结构的关键改进外，由于其较小的尺寸，这些纳米盘还能够研究膜蛋白与其（可溶性）伴侣蛋白之间的相互作用，而不会受到去污剂的影响，这些去污剂可能会破坏生物学上相关的相互作用。
