Chao Luomeng, Yang Yuxia
College of Animal Science and Technology, Inner Mongolia MINZU University, Tongliao 028000, China.
College of Computer Science and Technology, Inner Mongolia MINZU University, Tongliao 028000, China.
Biology (Basel). 2025 Mar 31;14(4):360. doi: 10.3390/biology14040360.
This study elucidates the structural determinants and optogenetic potential of Archaerhodopsin AR, a proton pump from sp. Ejinoor isolated from Inner Mongolian salt lakes. Through heterologous expression in BL21 (DE3) and integrative biophysical analyses, we demonstrate that AR adopts a stable trimeric architecture (129 kDa) with detergent-binding characteristics mirroring bacteriorhodopsin (BR); however, it exhibits a 10 nm bathochromic spectral shift (λmax = 550 nm) and elevated proton affinity (Asp-95 pKa = 3.5 vs. BR Asp-85 pKa = 2.6), indicative of evolutionary optimization in its retinal-binding electrostatic microenvironment. Kinetic profiling reveals AR's prolonged photocycle (100 ms vs. BR's 11 ms), marked by rapid M-state decay (3.3 ms) and extended dark-adaptation half-life (160 min), a bistable behavior attributed to enhanced hydrogen bond persistence (80%) and reduced conformational entropy (RMSD = 2.0 Å). Functional assays confirm light-driven proton extrusion (0.1 ng H⁺/mg·s) with DCCD-amplified flux (0.3 ng H⁺/mg·s) and ATP synthesis (0.3 nmol/mg·s), underscoring its synergy with H⁺-ATPase. Phylogenetic and structural analyses reveal 95% homology with AR4 and conservation of 11 proton-wire residues, despite divergent Trp/Tyr/Ser networks that redefine chromophore stabilization. AlphaFold-predicted models (TM-score > 0.92) and molecular docking identify superior retinoid-binding affinity (ΔG = -12.27 kcal/mol), while spectral specificity (550-560 nm) and acid-stable photoresponse highlight its adaptability for low-irradiance neuromodulation. These findings position AR as a precision optogenetic tool, circumventing spectral overlap with excitatory opsins and enabling sustained hyperpolarization with minimized phototoxicity. By bridging microbial energetics and optobioengineering, this work expands the archaeal rhodopsin toolkit and provides a blueprint for designing wavelength-optimized photoregulatory systems.
本研究阐明了嗜盐古紫质AR的结构决定因素和光遗传学潜力，AR是一种从内蒙古盐湖分离的嗜盐碱红菌属的质子泵。通过在BL21(DE3)中的异源表达和综合生物物理分析，我们证明AR采用稳定的三聚体结构(129 kDa)，其洗涤剂结合特性与细菌视紫红质(BR)相似；然而，它表现出10 nm的红移光谱(λmax = 550 nm)和更高的质子亲和力(Asp-95 pKa = 3.5，而BR的Asp-85 pKa = 2.6)，这表明其视黄醛结合静电微环境中的进化优化。动力学分析揭示了AR延长的光循环(100 ms，而BR为11 ms)，其特征是快速的M态衰减(3.3 ms)和延长的暗适应半衰期(160分钟)，这种双稳态行为归因于增强的氢键持久性(80%)和降低的构象熵(RMSD = 2.0 Å)。功能测定证实了光驱动的质子外排(0.1 ng H⁺/mg·s)，DCCD增强的通量(0.3 ng H⁺/mg·s)和ATP合成(0.3 nmol/mg·s)，强调了其与H⁺-ATP酶的协同作用。系统发育和结构分析表明，尽管Trp/Tyr/Ser网络不同，重新定义了发色团的稳定性，但与AR4有95%的同源性，并且保留了11个质子线残基。AlphaFold预测模型(TM分数> 0.92)和分子对接确定了更高的类视黄醇结合亲和力(ΔG = -12.27 kcal/mol)，而光谱特异性(550-560 nm)和酸稳定的光反应突出了其对低辐照神经调节的适应性。这些发现将AR定位为一种精确的光遗传学工具，避免了与兴奋性视蛋白的光谱重叠，并能够以最小的光毒性实现持续的超极化。通过将微生物能量学与光生物工程联系起来，这项工作扩展了古菌视紫红质工具包，并为设计波长优化的光调节系统提供了蓝图。
