Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, 07745, Jena, Germany.
Center for Molecular Biomedicine, Institute for Molecular Cell Biology, Jena University Hospital, Hans-Knöll-Str. 2, 07745, Jena, Germany.
Talanta. 2022 Jun 1;243:123332. doi: 10.1016/j.talanta.2022.123332. Epub 2022 Mar 3.
Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with ∼400 and ∼470 nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488 nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.
蛋氨酸氧化是一种可逆的翻译后蛋白质修饰，影响蛋白质功能，并与衰老和退行性疾病有关。然而，在活细胞或生物体中检测到积累的蛋氨酸氧化尚未实现。在这里，我们引入了一种基于超折叠绿色荧光蛋白（sfGFP）的蛋氨酸氧化的遗传编码探针（GEPMO），作为蛋氨酸氧化的特异性、多功能和综合传感器。在 sfGFP 中，将这个特定的蛋氨酸置于位置 147，当用 ∼400 和 ∼470nm 光激发时，该蛋氨酸的氧化为蛋氨酸亚砜会导致比率荧光变化。该传感器的表达强度和均一性适合于活细胞成像以及使用标准激光波长（405/488nm）的荧光激活细胞分选（FACS）实验。在哺乳动物细胞和酿酒酵母中表达时，该传感器蛋白以整合的方式忠实地报告蛋氨酸氧化的状态。针对膜和线粒体的变体提供了蛋氨酸氧化的亚细胞分辨率，例如通过内源原卟啉 IX 的照明报告特定位置的氧化。
