A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell
A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell
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监测活细胞中 LptD 中间体与 LptE 组装的光交联方法
DOI：
10.1007/978-1-0716-2581-1_7
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Akiyama Yoshinori
中科院分区：
文献类型：
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作者：
Miyazaki Ryoji;Mori Hiroyuki;Akiyama Yoshinori
阐明活细胞中蛋白质的动态行为对于理解生物过程的生理作用是极其重要的。利用光反应性非天然氨基酸的体内定位光交联法能够以高空间分辨率在体内分析蛋白质相互作用。最近，通过改进光交联技术，我们发展了分析新合成蛋白质动态相互作用的“Pixie”方法。在这里，我们描述了“Pixie”方法的详细方案及其在分析脂多糖转位组分、β桶外膜蛋白LptD和脂蛋白LptE的组装过程中的应用。
Elucidating the dynamic behavior of proteins in living cells is extremely important for understanding the physiological roles of biological processes. The site-specific in vivo photo-crosslinking approach using a photoreactive unnatural amino acid enables the analysis of protein interactions with high spatial resolution in vivo. Recently, by improving the photo-crosslinking technique, we developed the “PiXie” method for the analysis of dynamic interactions of newly synthesized proteins. Here, we describe the detailed protocols of the “PiXie” method and its application to the analysis of the assembly processes of the lipopolysaccharide translocon components, a β-barrel outer membrane protein, LptD, and a lipoprotein, LptE.