Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
The Institute for Solid State Physics, University of Tokyo, Kashiwa, Chiba, 277-8581, Japan.
Sci Rep. 2019 Aug 2;9(1):11283. doi: 10.1038/s41598-019-47445-5.
Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR's hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR's oligomerization state using double electron-electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.
绿硫菌视紫红质（GR）是一种能够潜在应用于光遗传学的蓝细菌质子泵。我们解析了 GR 的晶体结构，发现它与来自盐红菌的同源质子泵，类视黄醛（XR），在整体上具有相似性。我们确定了 GR 的跨膜结构域中的氢键网络以及跨膜螺旋的胞外侧相对于 XR 的相对位移的独特结构特征。通过拉曼光谱和闪光光解，我们发现晶体中的 GR 存在一种状态，其展示出与在脂质中观察到的功能形式相似的视黄醛构象和光化学循环。基于 GR 的晶体结构，我们选择了一个位点进行自旋标记，以使用双电子-电子共振（DEER）光谱确定 GR 的寡聚状态，并证明了 GR 的 pH 依赖性五聚体形成。GR 结构的确定及其五聚化倾向使我们能够揭示常见于光驱动细菌泵中的结构基序（延伸螺旋、3-ω基序和翻转的 B-C 环）在寡聚体形成中的作用。在这里，我们提出了一个新概念，根据它们的寡聚化倾向和这些结构决定因素之间的关系对这些泵进行分类。
