期刊
BIO-PROTOCOL
卷 11, 期 16, 页码 -出版社
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.4129
关键词
Protein translocation; Sec translocon; SecA; SecYEG; Protein transport; Signal sequence; Kinetics
类别
资金
- National Institutes of Health [5R21AI133514, 5R01GM121567, 5T32GM007231]
- Pew Charitable Trusts (Pew Biomedical Scholars Program)
This study presents a method for real-time measurement of protein translocation, allowing high-resolution kinetics of the actual translocation process to be resolved. By developing a detailed kinetic model, detailed mechanistic analyses of Sec-dependent protein translocation can be conducted.
The Sec translocon, consisting of a heterotrimeric transmembrane channel (SecYEG) and an associated ATPase (SecA), catalyzes the export of unfolded proteins from the cytosol in bacteria. Kinetically resolving protein translocation at high resolution yields mechanistic insight into the process. Translocation is typically followed by measuring the protection of proteins transported into lipid vesicles, which only allows visualization of translocation after it has already been completed and limits time resolution. Here, we describe the implementation of an assay for measuring translocation in real-time. By priming the reconstituted translocon with suitably engineered substrate proteins, the kinetics of the actual translocation process can be resolved at high resolution. To analyze translocation kinetics, we developed a detailed kinetic model of the process that includes on-pathway and off-pathway processes. Together, this experimental protocol and model permit detailed mechanistic analyses of Sec-dependent protein translocation. [GRAPHICS] .
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据