Combination of ¹⁵N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE.
Combination of ¹⁵N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE.
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DOI：
10.1007/s10858-013-9724-z
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发表时间：
2013-04
影响因子：
2.7
通讯作者：
Traaseth NJ
中科院分区：
文献类型：
--
作者：
Banigan JR;Gayen A;Traaseth NJ
魔角自旋（MAS）固态核磁共振光谱已成为表征膜蛋白结构和动力学的一种可行方法。然而，均匀标记样品的光谱分辨率经常受到初级序列冗余和螺旋二级结构存在的影响，导致大量共振重叠。简化光谱以获得明确的特定位点分配的能力是结构确定的主要瓶颈。为了解决这个问题，我们使用了15N反向标记和余辉光谱技术的组合，显着提高了在拥挤的光谱中分辨峰的能力。这是用多聚膜蛋白EmrE证明的，EmrE是一种参与多药耐药的外排泵。15N反向标记氨基酸之前的残基使用3D NCOCX余辉实验成像，之后的残基使用频率选择脱相实验记录。我们的方法减少了光谱拥塞，并提供了一种敏感的方法来获得膜蛋白的化学位移分配，在没有高分辨率结构可用的情况下。该方法可广泛应用于功能性脂质双分子层环境中其他多聚膜蛋白的研究。
Magic-angle-spinning (MAS) solid-state NMR spectroscopy has emerged as a viable method to characterize membrane protein structure and dynamics. Nevertheless, the spectral resolution for uniformly labeled samples is often compromised by redundancy of the primary sequence and the presence of helical secondary structure that results in substantial resonance overlap. The ability to simplify the spectrum in order to obtain unambiguous site-specific assignments is a major bottleneck for structure determination. To address this problem, we used a combination of 15N reverse labeling and afterglow spectroscopic techniques that dramatically improved the ability to resolve peaks in a crowded spectrum. This was demonstrated using the polytopic membrane protein EmrE, an efflux pump involved in multidrug resistance. Residues preceding the 15N reverse labeled amino acid were imaged using a 3D NCOCX afterglow experiment and those following were recorded using a frequency-selective dephasing experiment. Our approach reduced the spectral congestion and provided a sensitive way to obtain chemical shift assignments for a membrane protein where no high-resolution structure is available. This MAS methodology is widely applicable to the study of other polytopic membrane proteins in functional lipid bilayer environments.
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