Table 2_Archaeal LOV domains from Lake Diamante: first functional characterization of a halo-adapted photoreceptor.docx

High-altitude Andean lakes (HAALs) represent polyextreme environments where diverse photoinduced processes have been documented. In this study, we investigated Light-Oxygen-Voltage (LOV) photoreceptors and identified 28 archaeal sequences from Lake Diamante, which were classified into two major groups (A and B), with three outliers showing unique structural features. Analysis of these sequences and their 3D models revealed hallmark adaptations to halophilic environments, including an abundance of surface acidic residues, an increased prevalence of arginine over lysine, and a greater density of salt bridges. The heterologous expression of a representative LOV domain, ALovD-1, demonstrated conserved photophysics between its dark- and light-adapted states, which was consistent with the slow cycling type. Importantly, ALovD-1 exhibited remarkable halophilic characteristics, maintaining photocycling functionality at salt concentrations as high as 3 M monovalent salts. This ability can be attributed to discrete structural changes, allowing adjustments in flavin interactions within its cavity under varying ionic strengths. Mutational studies of key residues (Y30F and Y48F) highlighted their roles in modulating flavin photophysic and revealed a stabilizing function for Y48 at low salt concentrations. These findings mark the first functional characterization of a canonical archaeal LOV domain, expanding our understanding of light sensing and protein adaptation in extremophiles.

安第斯高原高海拔湖泊（High-altitude Andean lakes, HAALs）属于多极端环境，已有研究记录了其中存在多样的光诱导过程。本研究针对光-氧-电压（Light-Oxygen-Voltage, LOV）光感受器展开探究，从迪亚曼特湖（Lake Diamante）中鉴定得到28条古菌序列，这些序列被划分为两大类群（A类与B类），另有3条离群序列展现出独特的结构特征。对这些序列及其三维结构模型的分析显示，其具备嗜盐环境适应性的标志性特征：包括表面酸性残基富集、精氨酸相对赖氨酸的占比提升，以及盐桥密度更高。代表性LOV结构域ALovD-1的异源表达实验证实，其暗适应态与光适应态之间具备保守的光物理特性，这与慢循环型光感受器的特征一致。尤为关键的是，ALovD-1展现出优异的嗜盐特性：在高达3 mol/L的单价盐浓度条件下，仍可维持光循环功能。该特性可归因于其存在离散的结构变化，使得在离子强度改变时，其结合口袋内的黄素相互作用可得到相应调控。对关键残基（Y30F与Y48F）的突变研究明确了其在调控黄素光物理特性中的作用，并揭示Y48在低盐浓度下具备稳定功能。本研究成果首次完成了典型古菌LOV结构域的功能表征，拓展了我们对极端微生物中光感知与蛋白质适应性的认知。