A comparative study of influenza A M2 protein conformations in DOPC/DOPS liposomes and in native E. coli membranes.
A comparative study of influenza A M2 protein conformations in DOPC/DOPS liposomes and in native E. coli membranes.
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DOPC/DOPS 脂质体和天然大肠杆菌膜中甲型流感 M2 蛋白构象的比较研究。
DOI：
10.1101/2024.01.08.574681
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Georgieva,ElkaR
中科院分区：
文献类型：
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作者：
Sanders,Griffin;Borbat,PeterP;Georgieva,ElkaR
我们将在 pH 7.4 下在 DOPC/DOPS 双层中重建的甲型流感 M2 (IAM2) 蛋白的跨膜结构域 (TMD) 构象与分离的大肠杆菌膜中的构象进行了比较，保留了其天然蛋白和脂质。 IAM2 是一种单程跨膜蛋白，已知可组装成同源四聚体质子通道。为了表示这个通道，我们制作了一个包含 IAM2 的 TMD 区域的构建体，两侧是近膜残基。位于双层极性区域的亮氨酸的单一半胱氨酸替代物 L43C 被顺磁性标记为甲硫基磺酸盐氮氧化物标签，用于 ESR（电子自旋共振）研究。我们使用连续波 (CW) ESR 和双电子-电子共振 (DEER) 光谱比较了 DOPC/DOPS 和天然大肠杆菌膜中的自旋标记 IAM2 的构象。总蛋白质与脂质摩尔比的范围为 1:230 至 1:10,400⩦ CW ESR 光谱对应于两种环境中近乎严格的极限自旋标记动力学。在所有情况下，DEER 数据都被重建为距离分布，显示 1.68 nm 和 2.37 nm 处的峰分辨率良好。峰距比为1.41±0.2，振幅比为2:1。这是人们对位于正方形角上的四个硝基氧自旋标签的预期，表明轴对称四聚体。使用应用于 NMR 分子结构 (PDB: 2L0J) 的分子建模软件对 DEER 数据进行距离建模，证实了 IAM2 四聚体 C 端出口孔的对称性和闭合状态，与 NMR 模型一致。因此，我们可以得出结论，IAM2 TMD 在模型和天然大肠杆菌膜中具有相似的构象，厚度和流动性相当，尽管大肠杆菌膜因其脂质多样性和丰富的整体膜蛋白和外周膜蛋白而变得复杂。
We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IAM2) protein reconstituted at pH 7.4 in DOPC/DOPS bilayers to those in isolated E. coli membranes, having preserved its native proteins and lipids. IAM2 is a single-pass transmembrane protein known to assemble into homo-tetrameric proton channel. To represent this channel, we made a construct containing the IAM2’s TMD region flanked by the juxtamembrane residues. The single cysteine substitute, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the ESR (electron spin resonance) study. We compared the conformations of the spin-labeled IAM2 residing in DOPC/DOPS and native E. coli membranes using continuous-wave (CW) ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400⩦ The CW ESR spectra corresponded to a nearly rigid limit spin label dynamics in both environments. In all cases, the DEER data were reconstructed into the distance distributions showing well-resolved peaks at 1.68 nm and 2.37 nm. The peak distance ratio was 1.41±0.2 and the amplitude ratio was 2:1. This is what one expects from four nitroxide spin-labels located at the corners of a square, indicative of an axially symmetric tetramer. Distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IAM2 tetramer in agreement with the NMR model. Thus, we can conclude that IAM2 TMD has similar conformations in model and native E. coli membranes of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.