Milikisiyants Sergey, Wang Shenlin, Munro Rachel A, Donohue Matthew, Ward Meaghan E, Bolton David, Brown Leonid S, Smirnova Tatyana I, Ladizhansky Vladimir, Smirnov Alex I
Department of Chemistry, College of Sciences, North Carolina State University, 2620 Yarbrough Dive, Raleigh, NC 27695-8204, USA.
Beijing Nuclear Magnetic Resonance Center and College of Chemistry and Molecular Engineering, Peking University, 5 Yiheyuan Road, Haidian, Beijing 100871, People's Republic of China, Beijing National Laboratory for Molecular Sciences (BNLMS), Beijing, People's Republic of China.
J Mol Biol. 2017 Jun 16;429(12):1903-1920. doi: 10.1016/j.jmb.2017.05.005. Epub 2017 May 10.
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 3-60Å 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.
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）光谱技术，对在脂质环境中重构的寡聚整合膜蛋白鱼腥藻感光视紫红质（ASR）的结构进行优化。这种联合方法的一个基本特征是，它在为ssNMR和DEER使用相同的样品制备方法（即突变、顺磁标记和在脂质双层中重构）的同时，提供跨越约3 - 60Å范围的结构距离限制。对DEER信号的多自旋效应进行直接建模，可确定寡聚顺序，并获得ASR三聚体亚基之间的长程DEER距离限制，用于优化ASR的ssNMR结构。与仅使用ssNMR数据确定的结构相比，ASR三聚体改进后的结构显示在单体间界面处螺旋和侧链堆积更紧密。与同源蛋白活性状态观察到的典型螺旋运动相比，优化程度很显著。我们使用互补的DEER和NMR测量来确定寡聚结构的联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，以及观察蛋白质寡聚体在药物、底物和辅因子结合时的结构变化。
