Department of Physics and Astronomy, University of Missouri-Columbia, Columbia, MO 65211, USA.
Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA.
Sci Adv. 2018 Oct 24;4(10):eaat8797. doi: 10.1126/sciadv.aat8797. eCollection 2018 Oct.
SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in . This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.
SecA 是一种关键的三磷酸腺苷酶，它驱动前体蛋白通过 SecYEG 易位通道进行转运。这个过程被认为受到 SecA 特定结构域构象变化的调节，但目前缺乏对这些变化的实时、真实空间测量。我们使用单分子原子力显微镜 (AFM) 可视化 SecA 的核苷酸依赖性构象和构象动力学。在特定核苷酸存在的情况下，观察到了不同的地形种群。在基础三磷酸腺苷 (ATP) 水解过程中的 AFM 研究揭示了在 ~100-ms 的时间尺度内，紧凑态和扩展态之间的快速、可逆转换。缺乏前体结合结构域 (PBD) 的 SecA 突变体有助于解释这一现象。此外，通过跟踪无机磷酸盐的释放，证实了用于 AFM 的 SecA 的生化活性。我们的结论是，ATP 驱动的动力学主要归因于 PBD 的运动，但在 ATP 水解循环的过渡态期间，SecA 的其他片段也对这种运动有贡献。
