反布朗电动势阱中FRET标记的F1-ATPase分子的亚基旋转
Subunit rotation in single FRET-labeled F1-ATPase hold in solution by an anti-Brownian electrokinetic trap
摘要：
FoF 1-ATP合酶催化三磷酸腺苷（ATP）的合成。F1部分可以从酶的膜包埋的Fo部分剥离。F1充当ATP水解酶，并且ATP水解与F1的γ亚基和γ亚基的逐步旋转相关。在过去的15年里，使用附着在表面上的单个F1部件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测γ的亚基旋转。或者，我们应用单分子Förster共振能量转移（FRET）监测亚基旋转的全酶FoF 1-ATP合酶在脂质体中重建。现在，我们的目标是延长观察时间的单一FRET标记的F1在溶液中使用的反布朗电动势陷阱（ABELtrap）的修改版本发明的A。E. Cohen和W. E.莫尔纳我们使用Monte Carlo模拟来揭示逐步FRET效率变化可以通过隐马尔可夫模型来分析，即使在预期由于ABELtrap的微流体引起的高背景计数率而导致的低信号背景比的限制下。
英文摘要：
FoF1-ATP synthase catalyzes the synthesis of adenosine triphosphate (ATP). The F1 portion can be stripped from the membrane-embedded Fo portion of the enzyme. F1 acts as an ATP hydrolyzing enzyme, and ATP hydrolysis is associated with stepwise rotation of the gamma and epsilon subunits of F1. This rotary motion was studied in great detail for the last 15 years using single F1 parts attached to surfaces. Subunit rotation of gamma was monitored by videomicroscopy of bound fluorescent actin filaments, nanobeads or nanorods, or single fluorophores. Alternatively, we applied single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the holoenzyme FoF1-ATP synthase which was reconstituted in liposomes. Now we aim to extend the observation times of single FRET-labeled F1 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.
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