Institute of Chemical Sciences and Engineering, National Centre of Competence in Research Chemical Biology, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
J Am Chem Soc. 2011 Oct 12;133(40):16235-42. doi: 10.1021/ja206915m. Epub 2011 Sep 16.
Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold. Our best Snifit displayed fluorescence intensity ratio changes on living cells significantly higher than any previously reported cell-surface-targeted fluorescent sensor protein. This work establishes a generally applicable and rational strategy for the generation of cell-surface-targeted fluorescent sensor proteins for metabolites of interest.
在理解信号转导和代谢途径方面的进展受到缺乏合适的传感器的阻碍，这些传感器可用于跟踪活细胞中的代谢物、第二信使和神经递质。在这里，我们引入了一类经过合理设计的半合成荧光传感器蛋白，称为 Snifits，用于测量哺乳动物细胞表面的代谢物浓度。功能性 Snifits 通过对遗传编码的蛋白质支架进行两次选择性化学标记反应，在活细胞上组装。我们最好的 Snifit 在活细胞上显示出的荧光强度比任何以前报道的细胞表面靶向荧光传感器蛋白都要高得多。这项工作为生成针对感兴趣代谢物的细胞表面靶向荧光传感器蛋白建立了一种普遍适用和合理的策略。
