Ueta Tetsuya, Kojima Keiichi, Hino Tomoya, Shibata Mikihiro, Nagano Shingo, Sudo Yuki
Dentistry and Pharmaceutical Sciences, Graduate School of Medicine, Okayama University, Okayama, Japan.
Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan; Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan.
Biophys J. 2020 Nov 3;119(9):1760-1770. doi: 10.1016/j.bpj.2020.09.026. Epub 2020 Sep 29.
The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as "micelles." The extracted protein is purified by column chromatography and then is often reconstituted into "liposomes" by removal of the detergent. The styrene-maleic acid ("SMA") copolymer spontaneously forms nanostructures containing lipids without detergent. In this study, we applied SMA to characterize two microbial rhodopsins, a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin (RxR), and an unstable one, Halobacterium salinarum sensory rhodopsin I (HsSRI), and evaluated their physicochemical properties in SMA lipid particles compared with rhodopsins in micelles and in liposomes. Those two rhodopsins were produced in Escherichia coli cells and were successfully extracted from the membrane by the addition of SMA (5 w/v %) without losing their visible color. Analysis by dynamic light scattering revealed that RxR in SMA lipid particles (RxR-SMA) formed a discoidal structure with a diameter of 54 nm, which was 10 times smaller than RxR in phosphatidylcholine liposomes. The small particle size of RxR-SMA allowed us to obtain scattering-less visible spectra with a high signal-to-noise ratio similar to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside. High-speed atomic force microscopy revealed that a single particle contained an average of 4.1 trimers of RxR (12.3 monomers). In addition, RxR-SMA showed a fast cyclic photoreaction (k = 13 s) comparable with RxR in phosphatidylcholine liposomes (17 s) but not to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside (0.59 s). By taking advantage of SMA, we determined the dissociation constant (K) of chloride for HsSRI as 34 mM. From these results, we conclude that SMA is a useful molecule forming a membrane-mimicking assembly for microbial rhodopsins having the advantages of detergents and liposomes.
膜嵌入蛋白视紫红质作为一种光感受器在生物体中广泛产生，具有多种光依赖的生物学功能。为了研究其分子特征，视紫红质通常通过合适的去污剂以“胶束”形式从细胞膜脂质中提取出来。提取的蛋白质通过柱色谱法纯化，然后通常通过去除去污剂再组装成“脂质体”。苯乙烯 - 马来酸（“SMA”）共聚物可在无去污剂的情况下自发形成包含脂质的纳米结构。在本研究中，我们应用SMA来表征两种微生物视紫红质，一种是热稳定的视紫红质，嗜热栖热放线菌视紫红质（RxR），另一种是不稳定的嗜盐菌盐生盐杆菌感官视紫红质I（HsSRI），并与胶束和脂质体中的视紫红质相比，评估了它们在SMA脂质颗粒中的物理化学性质。这两种视紫红质在大肠杆菌细胞中产生，并通过添加SMA（5 w/v%）成功地从膜中提取出来，且未失去其可见颜色。动态光散射分析表明，SMA脂质颗粒中的RxR（RxR - SMA）形成了直径为54 nm的盘状结构，这比磷脂酰胆碱脂质体中的RxR小10倍。RxR - SMA的小颗粒尺寸使我们能够获得类似于由正十二烷基 - β - D - 麦芽糖苷组成的去污剂胶束中的RxR的、具有高信噪比的无散射可见光谱。高速原子力显微镜显示，单个颗粒平均包含4.1个RxR三聚体（12.3个单体）。此外，RxR - SMA表现出快速的循环光反应（k = 13 s），与磷脂酰胆碱脂质体中的RxR（17 s）相当，但与由正十二烷基 - β - D - 麦芽糖苷组成的去污剂胶束中的RxR（0.59 s）不同。利用SMA，我们确定了HsSRI的氯离子解离常数（K）为34 mM。从这些结果中，我们得出结论，SMA是一种有用的分子，它为微生物视紫红质形成了一种模拟膜的组装体，具有去污剂和脂质体的优点。
