Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is a powerful technique to determine the topology of membrane proteins in oriented lipid bilayers. Separated local field (SLF) experiments are central to this technique as they provide first-order orientational restraints, i.e., dipolar couplings and anisotropic chemical shifts. Despite the use of low-E (or E-free) probes, the heat generated during the execution of 2D and 3D SLF pulse sequences causes sizeable line-shape distortions. Here, we propose a new heat-compensated SE-SAMPI4 (hcSE-SAMPI4) pulse sequence that holds the temperature constant for the duration of the experiment. This modification of the SE-SAMPI4 results in sharper and more intense resonances without line-shape distortions. The spectral improvements are even more apparent when paramagnetic relaxation agents are used to speed up data collection. We tested the hcSE-SAMPI4 pulse sequence on a single-span membrane protein, sarcolipin (SLN), reconstituted in magnetically aligned lipid bicelles. In addition to eliminating peak distortions, the hcSE-SAMPI4 experiment increased the average signal-to-noise ratio by 20% with respect to the original SE-SAMPI4.

中文翻译：

---

通过热补偿分离局部场实验提高定向膜蛋白光谱的质量

定向样品固态NMR（OS-ssNMR）光谱是一种确定定向脂质双层中膜蛋白拓扑结构的强大技术。分离局部场（SLF）实验对该技术至关重要，因为它们提供了一级定向约束，即偶极耦合和各向异性化学位移。尽管使用了低E（或无E）探针，但在执行2D和3D SLF脉冲序列期间产生的热量仍会导致较大的线形失真。在这里，我们提出了一种新的热补偿SE-SAMPI4（hcSE-SAMPI4）脉冲序列，该脉冲序列在实验过程中保持温度恒定。SE-SAMPI4的这种修改可产生更清晰，更强烈的共振，而不会出现线形失真。当使用顺磁性松弛剂加速数据收集时，光谱的改善甚至更加明显。我们测试了hcSE-SAMPI4脉冲序列在重构为磁性排列的脂质双胞胎中的单跨膜蛋白肌钙蛋白（SLN）上的表达。除了消除峰值失真外，hcSE-SAMPI4实验还使原始SE-SAMPI4的平均信噪比提高了20％。