Di Paolo Diana, Berry Richard M
Cambridge Healthcare Research, St John's Innovation Centre, CB40WS, Cambridge, UK.
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK.
Methods Mol Biol. 2018;1729:233-246. doi: 10.1007/978-1-4939-7577-8_19.
For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates. Electroporation allows for internalization of purified CheY proteins labeled with organic dyes into E. coli cells in controllable concentrations. Using fluorescence video microscopy, it is possible to observe single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.
在过去的20年里，基因融合荧光蛋白（FPs）的使用极大地促进了对大肠杆菌趋化信号的研究，包括响应调节蛋白CheY的激活及其与鞭毛马达的相互作用。然而，这种方法存在许多生物学和生物物理方面的局限性。例如，当与体积较大的荧光蛋白融合时，并非所有融合蛋白都具有完全功能，该荧光蛋白的分子量可能与其融合的对应物相似。荧光蛋白可能会干扰蛋白质的天然相互作用，并且它们的发色团亮度低、光稳定性差且光漂白速率快。电穿孔可使标记有有机染料的纯化CheY蛋白以可控浓度内化到大肠杆菌细胞中。使用荧光视频显微镜，可以实时观察单个CheY分子在细胞内扩散并与传感簇和鞭毛马达相互作用。
