School of Life Science, Westlake University, Hangzhou, Zhejiang, China.
Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China.
Commun Biol. 2024 Oct 23;7(1):1375. doi: 10.1038/s42003-024-07020-9.
Genetically encoded fluorescent biosensors became indispensable tools for biological research, enabling real-time observation of physiological processes in live cells. Recent protein engineering efforts have resulted in the generation of a large variety of fluorescent biosensors for a wide range of biologically relevant processes, from small ions to enzymatic activity and signaling pathways. However, biosensors for imaging sulfate ions, the fourth most abundant physiological anion, in mammalian cells are still lacking. Here, we report the development and characterization of a green fluorescent biosensor for sulfate named Thyone. Thyone, derived through structure-guided design from bright green fluorescent protein mNeonGreen, exhibited a large negative fluorescence response upon subsecond association with sulfate anion with an affinity of 11 mM in mammalian cells. By integrating mutagenesis analyses with molecular dynamics simulations, we elucidated the molecular mechanism of sulfate binding and revealed key amino acid residues responsible for sulfate sensitivity. High anion selectivity and sensitivity of Thyone allowed for imaging of sulfate anion transients mediated by sulfate transporter heterologously expressed in cultured mammalian cells. We believe that Thyone will find a broad application for assaying the sulfate transport in mammalian cells via anion transporters and exchangers.
基因编码荧光生物传感器已成为生物学研究不可或缺的工具，使人们能够实时观察活细胞中的生理过程。最近的蛋白质工程努力产生了大量用于各种生物学相关过程的荧光生物传感器，从小离子到酶活性和信号通路。然而，用于哺乳动物细胞中成像硫酸盐离子（第四大丰富的生理阴离子）的生物传感器仍然缺乏。在这里，我们报告了一种名为 Thyone 的绿色荧光硫酸盐生物传感器的开发和特性。Thyone 通过结构引导设计从亮绿色荧光蛋白 mNeonGreen 衍生而来，在与硫酸盐阴离子结合后的亚秒内表现出大的负荧光响应，在哺乳动物细胞中的亲和力为 11mM。通过将突变分析与分子动力学模拟相结合，我们阐明了硫酸盐结合的分子机制，并揭示了负责硫酸盐敏感性的关键氨基酸残基。Thyone 具有高阴离子选择性和灵敏度，可用于成像通过在培养的哺乳动物细胞中异源表达的硫酸盐转运体介导的硫酸盐阴离子瞬变。我们相信 Thyone 将通过阴离子转运体和交换体广泛应用于检测哺乳动物细胞中的硫酸盐转运。
