Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States.
Prog Nucl Magn Reson Spectrosc. 2013 Nov;75:50-68. doi: 10.1016/j.pnmrs.2013.07.004. Epub 2013 Aug 12.
Oriented solid-state NMR (O-ssNMR) spectroscopy is a major technique for the high-resolution analysis of the structure and topology of transmembrane proteins in native-like environments. Unlike magic angle spinning (MAS) techniques, O-ssNMR spectroscopy requires membrane protein preparations that are uniformly oriented (mechanically or magnetically) so that anisotropic NMR parameters, such as dipolar and chemical shift interactions, can be measured to determine structure and orientation of membrane proteins in lipid bilayers. Traditional sample preparations involving mechanically aligned lipids often result in short relaxation times which broaden the (15)N resonances and encumber the manipulation of nuclear spin coherences. The introduction of lipid bicelles as membrane mimicking systems has changed this scenario, and the more favorable relaxation properties of membrane protein (15)N and (13)C resonances make it possible to develop new, more elaborate pulse sequences for higher spectral resolution and sensitivity. Here, we describe our recent progress in the optimization of O-ssNMR pulse sequences. We explain the theory behind these experiments, demonstrate their application to small and medium size proteins, and describe the technical details for setting up these new experiments on the new generation of NMR spectrometers.
定向固态核磁共振（O-ssNMR）光谱学是一种用于在类似天然环境中对跨膜蛋白的结构和拓扑进行高分辨率分析的主要技术。与魔角旋转（MAS）技术不同，O-ssNMR 光谱学需要均匀定向（机械或磁场）的膜蛋白制剂，以便可以测量各向异性 NMR 参数，如偶极和化学位移相互作用，从而确定脂质双层中膜蛋白的结构和取向。涉及机械排列脂质的传统样品制备通常会导致较短的弛豫时间，从而使（15）N 共振变宽，并妨碍核自旋相干的操纵。脂质双分子层作为膜模拟系统的引入改变了这种情况，并且膜蛋白（15）N 和（13）C 共振的更有利的弛豫特性使得有可能开发用于更高光谱分辨率和灵敏度的新的、更复杂的脉冲序列。在这里，我们描述了我们在优化 O-ssNMR 脉冲序列方面的最新进展。我们解释了这些实验背后的理论，展示了它们在小和中等大小蛋白质中的应用，并描述了在新一代 NMR 光谱仪上设置这些新实验的技术细节。
