A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes
A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes
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DOI：
10.1016/j.jmb.2019.03.007
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发表时间：
2019-04-05
影响因子：
5.6
通讯作者：
Collinson, Ian
中科院分区：
文献类型：
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作者：
Pereira, Goncalo C.;Allen, William J.;Collinson, Ian
蛋白质转运是生物学中的一个基本过程。我们对这一过程的理解存在重大差距，这是由于易位测定的灵敏度差、时间分辨率低和不可重复性。为了解决这个问题，我们应用NanoLuc分裂荧光素酶来产生一种测量蛋白质转运的新策略。该系统将数据收集的时间尺度从几天减少到几分钟，并允许以秒级的时间分辨率连续采集，产生适合于机理阐明和数学拟合的动力学参数。为了证明其多功能性，我们在体外和体内实施并验证了细菌Sec系统和线粒体蛋白输入装置的测定。总的来说，这项技术代表了向前迈出的重要一步，为蛋白质易位的基本机制研究和抑制剂（药物）筛选提供了一个强大的新工具，其强度和严谨性是经典方法无法达到的。(C)2019作者爱思唯尔有限公司出版
Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods. (C) 2019 The Authors. Published by Elsevier Ltd.