Single-molecule FRET dynamics of molecular motors in an ABEL trap.
Single-molecule FRET dynamics of molecular motors in an ABEL trap.
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ABEL 陷阱中分子马达的单分子 FRET 动力学。
DOI：
10.1016/j.ymeth.2021.01.012
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Dienerowitz M
中科院分区：
文献类型：
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作者：
Dienerowitz M
分子马达的单分子Förster共振能量转移（smFRET）同时在高时间和空间分辨率下提供了对其动力学和构象变化的变革性见解。然而，这种FRET研究的一个关键挑战是观察分子的作用足够长的时间，而不限制其自然功能。反布朗电动势阱（ABEL阱）着手将联合收割机smFRET与分子限制结合，以使观察时间长达数秒，同时消除所讨论的分子的束缚表面附着的任何要求。此外，ABEL陷阱选择性捕获FRET活性分子的固有能力加速了数据采集过程。在这项工作中，我们研究了ABEL陷阱在分子马达Rep上进行延长时间尺度smFRET测量的能力，这对于在推进DNA复制机制之前去除蛋白质块和重新启动停滞的DNA复制至关重要。我们能够监测单个Rep分子长达6秒，具有亚毫秒的时间分辨率，在观察时间内捕获多个构象转换事件。在这里，我们提供了一个循序渐进的指导合理的设计，建设和实施的ABEL陷阱的smFRET检测Repin体外。我们包括如何在陷阱网站建模的电势，并使用隐马尔可夫分析的smFRET轨迹的细节。
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 seconds 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 Repin vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.