Koroloff Sophie N, Tesch Deanna M, Awosanya Emmanuel O, Nevzorov Alexander A
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA.
Shaw University, 118 E. South Street, Raleigh, NC, 27601, USA.
J Biomol NMR. 2017 Feb;67(2):135-144. doi: 10.1007/s10858-017-0090-0. Epub 2017 Feb 15.
Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.
取向样品固态核磁共振所采用的多维分离局部场和自旋交换实验对于重构于宏观排列脂质双层中的膜蛋白的结构测定和光谱归属至关重要。然而，这些实验通常需要大量扫描才能建立自旋间相关性。在此我们表明，对于重构于磁取向双分子层中的Pf1外壳蛋白，其法线与主磁场平行或垂直，优化的重复交叉极化（REP-CP）和膜嵌入自由基的组合能够将信噪比提高2.4至3.0倍。值得注意的是，在自由基与蛋白比例为2:1时，光谱分辨率不受影响。作为该方法的一个示例，已确定了平行双分子层中Pf1外壳蛋白的光谱归属。当应用于含有较少量蛋白质的样品和三维实验时，所提出的方法将推动取向样品核磁共振技术的应用。
