Department of Chemistry, Graduate School of Science , Osaka University , 1-1 Machikaneyama , Toyonaka , Osaka 560-0043 , Japan.
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University , 1-1-1 Tsushima-naka , Kita-ku, Okayama 700-8530 , Japan.
J Phys Chem B. 2018 Jul 12;122(27):6945-6953. doi: 10.1021/acs.jpcb.8b04894. Epub 2018 Jun 27.
Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.
嗜热视紫红质（TR）是一种来自极端嗜热菌 Thermus thermophilus JL-18 的光驱动质子泵。以前使用去污剂溶解 TR 的研究表明，该蛋白质表现出高热稳定性，在室温下形成三聚体，但在生理温度（75°C）下孵育时不可逆地解离为单体。在本研究中，我们使用共振拉曼（RR）光谱、固态 NMR 光谱和高速原子力显微镜分析了 TR 在脂质环境中的寡聚结构。获得的光谱和微观图像表明，TR 在脂质环境中采用五聚体形式，并且这种组装在生理温度下稳定，与蛋白质在溶解状态下的行为形成对比。这些结果表明，TR 的寡聚组装在脂质环境中的热稳定性高于去污剂胶束。观察到的 RR 光谱还表明，视黄醛发色团通过质子化的席夫碱与内部水分子强烈氢键结合，这是质子泵视紫红质的特征。获得的数据强烈表明，TR 在极端嗜热菌 T. thermophilus JL-18 中的生理温度下以五聚体形式发挥作用。我们利用溶解 TR 的单体形式的高热稳定性，并在此报告了第一个微生物视紫红质单体形式的 RR 光谱。观察到的 RR 光谱表明，TR 的单体化改变了发色团结构：多烯链的键交替和质子化的席夫碱的氢键强度发生变化。本研究揭示了 TR 在脂质环境中的寡聚组装的高热稳定性，并表明使用嵌入脂质膜中的 TR 阐明其功能机制的重要性。
