Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane‐embedded translocon SecYEG that interacts with the secretion‐dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single‐molecule level. Silane‐based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide‐free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm²·s⁻¹. Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.

中文翻译：

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SecYEG转运子动力学和相互作用的单分子分析

蛋白质易位并插入细菌细胞质膜是由Sec机制介导的基本过程。核心机制是由膜嵌入的转录子SecYEG组成，该膜与分泌专用的ATPase SecA相互作用并翻译核糖体。尽管简单性和对系统的可用结构见解，但已提出了多种分子机制和功能动力学。在这里，我们采用全内反射荧光显微镜研究了单分子水平上SecYEG易位子在支持的脂质双层中的低聚状态和扩散。基于硅烷的涂层可确保脂质和双层内重组脂质体的迁移。亮度分析表明，约。70％的转座子是单体的。核糖体结合后，转座子仍以单体形式存在，但在无核苷酸的SecA存在下发生部分寡聚。脂质双层中SecYEG的各个轨迹显示了扩散的动态异质性，因为易位子通常在慢速和快速迁移模式之间切换，相应的扩散系数为0.03和0.7 µm² ·s ^-1。与SecA ATPase的相互作用对侧向运动影响较小，而结合的核糖体：新生链复合物则显着阻碍了单个易位子的扩散。值得注意的是，Translocon：核糖体复合物的迁移率不受Ficoll PM 70调节的溶剂粘度或大分子拥挤的影响，因此很大程度上取决于脂质双层内部和界面处的相互作用。我们建议，SecYEG的复杂流动性来自转运蛋白和蛋白质：脂质相互作用的构象动力学。