Kurihara Marie, Thiel Vera, Takahashi Hirona, Kojima Keiichi, Ward David M, Bryant Donald A, Sakai Makoto, Yoshizawa Susumu, Sudo Yuki
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University.
Department of Biological Sciences, Tokyo Metropolitan University.
Chem Pharm Bull (Tokyo). 2023;71(2):154-164. doi: 10.1248/cpb.c22-00774.
Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na pump function across the cellular membrane. Thus, we named the protein Bellilinea Na-pumping rhodopsin, BeNaR. Of note, its Na-pumping activity is significantly greater than that of the known Na-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na-pumping activity. From the results, we propose a model for the outward Na-pumping mechanism of BeNaR. The efficient Na-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.
视紫红质是一种跨膜蛋白，含有视黄醛发色团，参与多种生物体中的光能转换和光信号转导。在本研究中，我们新鉴定并表征了一种来自嗜热细菌Bellilinea sp.的视紫红质。表达该视紫红质的重组大肠杆菌细胞仅在存在钠离子（Na）的情况下才表现出光诱导的培养基碱化，并且通过添加质子载体可增强碱化信号，这表明其具有跨细胞膜的外向钠泵功能。因此，我们将该蛋白命名为Bellilinea钠泵视紫红质，即BeNaR。值得注意的是，其钠泵活性明显高于已知的钠泵视紫红质KR2。我们进一步对其光化学性质进行了如下表征：（i）可见光谱和高效液相色谱显示，BeNaR在524 nm处有最大吸收峰，主要（>96%）为全反式视黄醛构象体。（ii）时间依赖性热变性实验表明，BeNaR具有高热稳定性。（iii）纳秒至毫秒时间域内的时间分辨闪光光解揭示了存在四种动力学上不同的光中间体，即K、L、M和O。（iv）突变分析表明，作为抗衡离子的Asp101和视黄醛周围的Asp230对钠泵活性至关重要。根据这些结果，我们提出了BeNaR外向钠泵机制的模型。BeNaR高效的钠泵活性及其高稳定性使其成为离子转运体和光遗传学工具的有用模型。
