Department of Physics and Astronomy, University of Missouri, Columbia, MO 65211, USA.
Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.
Sci Adv. 2019 Jun 12;5(6):eaav9404. doi: 10.1126/sciadv.aav9404. eCollection 2019 Jun.
exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.
它通过包含 SecA 和转位通道 SecYEG 的易位子来输出蛋白质。活性易位子的结构变化是一般分泌系统功能的基础，但直接可视化动力学一直具有挑战性。我们使用原子力显微镜 (AFM) 研究了脂质双层中活性易位子作为前体蛋白种类、核苷酸种类和转位阶段的功能。从几乎相同的初始状态开始，与结合了外膜蛋白 A 的前体相比，SecA 更容易从 SecYEG 中解离，而与结合了半乳糖结合蛋白的前体相比则更不容易。对于仍然与转位通道结合的 SecA，其四级结构随核苷酸而变化，主要是腺苷二磷酸 (ADP) 和三磷酸腺苷，SecA 单体则与过渡态类似物 ADP-AlF 结合。在 AFM 成像时间尺度上，转位酶的构象表现出与前体依赖性的差异。在近天然条件下获得的数据表明，转位过程随前体种类而变化。
