Single-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap's inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. In this work we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with sub-millisecond time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

中文翻译：

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ABEL阱中分子电动机的单分子FRET动力学

分子电动机的单分子Förster共振能量转移（smFRET）可同时以高时间和空间分辨率提供有关其动力学和构象变化的变革性见解。然而，这种FRET研究的关键挑战是观察一个作用足够长的分子而又不限制其天然功能。Anti-Brownian电动诱捕器（ABEL诱捕器）着手将smFRET与分子限制相结合，以使观察时间长达数秒，同时消除了对所述分子的束缚表面附着的任何要求。此外，ABEL阱固有的选择性捕获FRET活性分子的能力可加快数据采集过程。在这项工作中，我们举例说明了ABEL捕集阱在分子电动机Rep上执行扩展的时标smFRET测量的功能，这对于在先进的DNA复制机制之前去除蛋白质块以及重新启动停滞的DNA复制至关重要。我们能够以亚毫秒级的时间分辨率监视长达6 s的单个Rep分子，从而在观察时间内捕获多个构象转换事件。在这里，我们为smFRET检测Rep的ABEL阱的合理设计，构建和实施提供了逐步指南。我们能够以亚毫秒级的时间分辨率监视长达6 s的单个Rep分子，从而在观察时间内捕获多个构象转换事件。在这里，我们为smFRET检测Rep的ABEL阱的合理设计，构建和实施提供了逐步指南。我们能够以亚毫秒级的时间分辨率监视长达6 s的单个Rep分子，从而在观察时间内捕获多个构象转换事件。在这里，我们为smFRET检测Rep的ABEL阱的合理设计，构建和实施提供了逐步指南。体外。我们将详细介绍如何对陷阱位置的电势建模以及如何使用smFRET轨迹的隐马尔可夫分析。