Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints
Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints
复制标题
结合固态 NMR 和长程 DEER 约束研究脂质双层环境中鱼腥藻感觉视紫红质的寡聚结构
DOI：
10.1016/j.jmb.2017.05.005
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发表时间：
2017-06-16
影响因子：
5.6
通讯作者：
Smirnov, Alex I.
中科院分区：
文献类型：
--
作者：
Milikisiyants, Sergey;Wang, Shenlin;Smirnov, Alex I.
膜蛋白的寡聚化在自然界中很常见。在这里，我们结合自旋标记双电子电子共振 (DEER) 和固态核磁共振 (ssNMR) 光谱来精炼在脂质环境中重构的寡聚整合膜蛋白鱼腥藻感觉视紫红质 (ASR) 的结构。这种组合方法的一个基本特征是，它提供了跨越约 3-60 埃范围的结构距离限制，同时对 ssNMR 和 DEER 使用相同的样品制备（即突变、顺磁标记和脂质双层重建）。对 DEER 信号的多自旋效应的直接建模可以确定寡聚顺序，并获得 ASR 三聚体亚基之间的远程 DEER 距离限制，用于细化 ASR 的 ssNMR 结构。与单独使用 ssNMR 数据确定的结构相比，ASR 三聚体的改进结构揭示了单体间界面处螺旋和侧链的更紧凑堆积。与观察到的同源蛋白质活性状态的典型螺旋运动相比，细化的程度是显着的。我们使用互补的 DEER 和 NMR 测量来确定寡聚结构的组合方法将广泛适用于可以引入顺磁标签的膜蛋白。这种方法可用于研究脂质膜组成对蛋白质寡聚化的影响，并观察药物、底物和辅因子结合时蛋白质寡聚物的结构变化。 (C) 2017 Elsevier Ltd. 保留所有权利。
Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3-60 angstrom while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding. (C) 2017 Elsevier Ltd. All rights reserved.