期刊
STRUCTURE
卷 27, 期 1, 页码 90-+出版社
CELL PRESS
DOI: 10.1016/j.str.2018.10.006
关键词
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资金
- Research Foundation Flanders (FWO) [G.0B49.15, G0683.15, G0A5817.N, ZW15_09 GOH6316N, G0C6814N RiMembR]
- Flemish government through long-term structural funding Methusalem CASAS2 [Meth/15/04]
- FWO/F.R.S. - FNRS Excellence of Science-EOS program [30550343]
- KU Leuven grant [C14/16/053]
- EU [FP7 KBBE.2013.3.6-02, 613877 StrepSynth]
- Agency for Innovation by Science and Technology in Flanders (IWT)
- RUN (KU Leuven) [RUN/16/001]
SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Forster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.
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