Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
ACS Sens. 2023 Aug 25;8(8):3014-3022. doi: 10.1021/acssensors.3c00484. Epub 2023 Jul 23.
Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.
基于荧光蛋白的基因编码 pH 传感器是用于成像与 pH 变化相关的细胞事件（如胞吐和胞吞作用）的有价值的工具。超亮 GFP（SEP）是一种广泛用于此类应用的 pH 敏感型绿色荧光蛋白（GFP）变体。在这里，我们报告了 Lime 的合理设计、开发、结构和应用，Lime 是一种改良的 SEP 变体，具有更高的荧光亮度和更大的 pH 敏感性。Lime 的 X 射线晶体结构支持了引导引入有益突变的机制原理。与 SEP 相比，Lime 可显著改善内吞作用和胞吐作用的成像。此外，Lime 及其变体在更广泛的应用中具有优势，包括检测突触释放和神经元电压变化。
