Department of Physics and Astronomy, University of Missouri, Columbia, Missouri, USA.
Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.
J Biol Chem. 2022 Oct;298(10):102412. doi: 10.1016/j.jbc.2022.102412. Epub 2022 Aug 22.
The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.
普遍分泌或 Sec 系统是大肠杆菌和所有真核生物细胞质中主要的蛋白质输出途径。膜蛋白复合物 SecDF 是一种易位子因子，可增强由 Sec 易位子（由易位子 SecYEG 和 ATP 酶 SecA 组成）驱动的多肽分泌，SecDF 被认为利用质子梯度有效地将前体蛋白从细胞质中拉入微周质。已经开发出工作模型来描述 SecDF 的结构和功能，但重要的机械问题仍未得到解答。原子力显微镜 (AFM) 是一种强大的技术，可用于研究单分子系统的动力学，包括近天然条件下的膜蛋白。AFM 的锋利尖端可直接进入膜外蛋白构象。在这里，我们在近天然的支持脂质双层中获取 AFM 图像和速度图（∼100 ms 分辨率）以可视化 SecDF 突起，并将实验数据与基于静态结构的模拟 AFM 图像进行比较。当单独研究时，SecDF 表现出接近脂质双层表面的稳定和紧凑构象，表明处于休息状态。有趣的是，在引入 SecYEG 后，我们观察到 SecDF 构象和构象动力学都发生了变化。与中间形式的 SecDF 相对应的周质突起的种群增加了九倍以上，这种中间形式的 SecDF 被认为在前体蛋白处理中具有活性。同时，我们的动力学测量结果表明，当易位子存在时，不同 SecDF 构象之间的转变速率增强。总之，这项工作提供了近天然条件下基础水平 SecDF 构象动力学的新视角。
