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首页> 外文会议>Single molecule spectroscopy and superresolution imaging VI >Subunit rotation in single FRET-labeled F_1-ATPase hold in solution by an anti-Brownian electrokinetic trap
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Subunit rotation in single FRET-labeled F_1-ATPase hold in solution by an anti-Brownian electrokinetic trap

机译：单个FRET标记的F_1-ATPase中的亚基旋转通过抗布朗电动势阱保持在溶液中

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摘要

F_0F_1-ATP synthase catalyzes the synthesis of adenosine triphosphate (ATP). The F_1, portion can be stripped from the membrane-embedded F_0 portion of the enzyme. F_1 acts as an ATP hydrolyzing enzyme, and ATP hydrolysis is associated with stepwise rotation of the γ and e subunits of F_1. This rotary motion was studied in great detail for the last 15 years using single F, parts attached to surfaces. Subunit rotation of γ was monitored by videomicroscopy of bound fluorescent actin filaments, nanobeads or nanorods, or single fluorophores. Alternatively, we applied single-molecule Forster resonance energy transfer (FRET) to monitor subunit rotation in the holoenzyme F_0F_1-ATP synthase which was reconstituted in liposomes. Now we aim to extend the observation times of single FRET-labeled F_1 in solution using a modified version of the anti-Brownian electrokinetic trap (ABELtrap) invented by A. E. Cohen and W. E. Moerner. We used Monte Carlo simulations to reveal that stepwise FRET efficiency changes can be analyzed by Hidden Markov Models even at the limit of a low signal-to-background ratio that was expected due to high background count rates caused by the microfluidics of the ABELtrap.
机译：F_0F_1-ATP合酶催化三磷酸腺苷（ATP）的合成。可以从酶的膜包埋的F_0部分剥离F_1部分。 F_1充当ATP水解酶，并且ATP水解与F_1的γ和e亚基的逐步旋转有关。在过去的15年中，使用单个F零件固定在表面上对这种旋转运动进行了详细的研究。通过结合的荧光肌动蛋白丝，纳米珠或纳米棒或单个荧光团的视频显微镜监控γ的亚基旋转。另外，我们应用了单分子Forster共振能量转移（FRET）来监测重组在脂质体中的全酶F_0F_1-ATP合酶中的亚基旋转。现在，我们的目的是使用A. E. Cohen和W. E. Moerner发明的抗布朗电动诱捕器（ABELtrap）的改进版本来延长溶液中单个FRET标记的F_1的观察时间。我们使用蒙特卡洛模拟来揭示，即使在由于ABELtrap的微流体导致的高背景计数率而预期的低信噪比的极限下，也可以通过Hidden Markov模型来分析FRET效率的逐步变化。

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  • 会议地点 San Francisco CA(US)
  • 作者

    Hendrik Sielaff; Thomas Heitkamp; Andrea Zappe; Nawid Zarrabi; Michael Boersch;

  • 作者单位

    Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University Jena, Nonnenplan 2-4,07743 Jena, Germany;

    Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University Jena, Nonnenplan 2-4,07743 Jena, Germany;

    3rd Institute of Physics, University of Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany;

    3rd Institute of Physics, University of Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany;

    Single-Molecule Microscopy Group, Jena University Hospital, Friedrich Schiller University Jena, Nonnenplan 2-4,07743 Jena, Germany, 3rd Institute of Physics, University of Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    F_1-ATPase; subunit rotation; single-molecule FRET; Hidden Markov Model; ABELtrap;

    机译：F_1-ATP酶；亚单元旋转单分子FRET;隐马尔可夫模型； ABEL陷阱;

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