Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles.
Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles.
复制标题
使用定向 Bicelles 的静态和偏魔角旋转固态核磁共振研究流感病毒 M2 蛋白的曲率诱导和膜定位。
DOI：
10.1021/acs.biochem.5b00127
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Hong,Mei
中科院分区：
文献类型：
--
作者：
Wang,Tuo;Hong,Mei
各种各样的膜蛋白诱导膜曲率的功能，因此，重要的是要开发新的方法，同时确定膜曲率和蛋白质结合位点的膜与多个曲率。我们介绍了固态核磁共振（NMR）方法的基础上磁定向bicelles和离魔角自旋（OMAS）测量膜曲率和混合曲率膜中的蛋白质的结合位点。我们在流感病毒M2蛋白上证明了这些方法，M2蛋白不仅充当质子通道，而且还介导病毒组装和膜断裂。研究了一种包含跨膜（TM）结构域和两亲性螺旋的M2肽M2（21-61），并与TM肽（M2 TM）进行了比较。磁性取向的1，2-二肉豆蔻酰-sn-甘油基-3-磷酸胆碱（DMPC）/1，2-二己酰-sn-甘油基-3-磷酸胆碱（DHPC）双胞的静态31 P NMR谱在M2（21-61）而非M2 TM存在下表现出与温度无关的各向同性化学位移，表明两亲性螺旋赋予产生高曲率相的能力。二维（2D）31 P光谱表明，这种高曲率相与DHPC双胞边缘，提示从宿主细胞的出芽病毒的结构。31 P-和13 C-检测的1H弛豫时间的脂质表明，大多数M2（21-61）是绑定到高曲率相。利用OMAS实验，我们分辨了具有相同头基的脂质的31 P信号，基于它们不同的化学位移各向异性。在此分辨率的基础上，2D 1H-31 P相关光谱显示M2（21-61）中的酰胺质子与双胞的DMPC信号相关，但与双胞的DHPC 31 P信号无关，表明小百分比的M2（21-61）分配到双胞的平面区域中。这些结果表明，两亲性螺旋诱导高膜曲率和定位的蛋白质到这个阶段，在良好的协议与蛋白质的膜断裂功能。这些基于双胞的弛豫和OMAS固态NMR技术通常适用于曲率诱导膜蛋白，例如参与膜运输、膜融合和细胞分裂的那些蛋白。
A wide variety of membrane proteins induce membrane curvature for function; thus, it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state nuclear magnetic resonance (NMR) methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21–61), was studied and compared with the TM peptide (M2TM). Static31P NMR spectra of magnetically oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21–61) but not M2TM, indicating that the amphipathic helix confers the ability to generate a high-curvature phase. Two-dimensional (2D)31P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell.31P- and13C-detected1H relaxation times of the lipids indicate that the majority of M2(21–61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the31P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. On the basis of this resolution, 2D1H–31P correlation spectra show that the amide protons in M2(21–61) correlate with the DMPC but not DHPC31P signal of the bicelle, indicating that a small percentage of M2(21–61) partitions into the planar region of the bicelles. These results show that the amphipathic helix induces high membrane curvature and localizes the protein to this phase, in good agreement with the membrane scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are generally applicable to curvature-inducing membrane proteins such as those involved in membrane trafficking, membrane fusion, and cell division.