Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695-8204, USA.
J Biomol NMR. 2012 Nov;54(3):307-16. doi: 10.1007/s10858-012-9673-y. Epub 2012 Sep 14.
Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the "shotgun" approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the (15)N chemical shift and (15)N-(1)H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an (15)N-(15)N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled "seed" spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B (1) fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.
定向样品核磁共振（OS-NMR）已成为在生理环境中确定膜蛋白结构的有力工具。然而，OS-NMR 中传统的基于“一枪打”策略的光谱解析方法虽然有效，但非常耗时耗力，因为它基于多个选择性标记样品的制备。在这里，我们展示了通过使用失谐哈特曼-哈恩条件下的自旋交换和最近的灵敏度增强 REP-CP 序列的组合，可以显著改善 Pf1 外壳蛋白在磁定向双胶束中重建的固态 NMR 光谱的光谱解析。该方法产生了 Pf1 外壳蛋白的二维自旋交换版本的 SAMPI4 谱，关联了 (15)N 化学位移和 (15)N-(1)H 偶极耦合，以及 (i，i±1)酰胺位点之间的自旋相关。将自旋交换的 SAMPI4 谱与原始的 SAMPI4 实验相结合，使得建立序列分配成为可能，并且该技术通常适用于其他单轴定向的膜蛋白。在分配过程中包含 (15)N-(15)N 相关谱有助于在自旋交换的 SAMPI4 实验中拥挤或不确定的谱区的峰之间建立相关性。值得注意的是，与传统方法不同，仅需要均匀标记的蛋白质样品即可进行光谱解析，可能只需要几个选择性标记的“种子”谱。还针对各种 B(1)场下失谐哈特曼-哈恩条件下稀散自旋之间的磁化转移进行了模拟。结果充分描述了建立交叉峰的最佳条件，从而消除了冗长的实验优化的需要。
