Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures.
Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures.
复制标题
生物正交标记能够对表达的气体囊泡纳米结构进行原位荧光成像。
DOI：
10.1021/acs.bioconjchem.3c00518
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发表时间：
2024
影响因子：
4.7
通讯作者：
Shapiro,MikhailG
中科院分区：
文献类型：
--
作者：
Schrunk,Erik;Dutka,Przemysław;Hurt,RobertC;Wu,Di;Shapiro,MikhailG
气体囊泡（GVs）是一种蛋白质纳米结构，与病毒样颗粒、胶囊、纳米笼和其他大分子组件一起，正在开发用于潜在的生物医学应用。为了促进这种发展，表征这些纳米结构的亚细胞组装和定位将是有价值的。然而，传统的荧光蛋白融合不能被gv的主要成分蛋白耐受，这使得光学显微镜成为一个挑战。在这里，我们介绍了一种使用生物正交标记FlAsH荧光可视化细胞内gv的方法，该标记与六氨基酸四胱氨酸（TC）标记反应后变为荧光。我们设计了GV亚基蛋白GvpA来显示TC标签，并表明携带TC标记GvpA的GV可以在HEK 293T细胞中成功组装并荧光可见。重要的是，这是通过用标记版本替换一小部分GvpA来实现的。我们利用标记的GV的荧光图像来研究GV在这些细胞内的大小和距离分布。这种生物正交和分数标记方法将使研究能够更好地了解gv，并可适用于类似的蛋白质纳米结构。
Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures’ subcellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs’ primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon reaction with the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.