Department of Microbiology and Molecular Genetics, University of California, Davis, California 95616, USA.
Nature. 2013 Aug 22;500(7463):482-5. doi: 10.1038/nature12333. Epub 2013 Jul 14.
Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.
单分子研究可以克服体相测量中异步和整体平均的复杂性，提供分子活动的机制见解，并揭示单个分子之间有趣的变化。将这些技术应用于大肠杆菌的 RecBCD 解旋酶已经解决了一些长期存在的差异，并为其酶促行为提供了其他无法获得的机制见解。奇怪的是，单个酶分子的 DNA 解旋速率变化很大，但这种异质性的起源尚不清楚。在这里，我们研究了这种行为的物理基础。尽管任何单个 RecBCD 分子在平均约 30000 步的时间内以恒定速率解开 DNA，但我们发现通过耗尽 Mg(2+)-ATP 暂时使单个酶-DNA 复合物停止，可以改变该酶在重新引入配体后随后解开 DNA 的速率。改变速率的分子比例与中断持续时间呈指数增长，半衰期约为 1 秒，这表明在分子被捕获的时间内发生了构象变化。在单个分子暂停后，速度是在整个酶-DNA 复合物的起始分布中找到的任何速度。我们认为，底物结合将酶稳定在决定每个 RecBCD 分子限速易位行为的许多平衡构象亚态之一中。每个稳定的亚态都可以持续稳定（约 1 分钟）解开一个 DNA 分子的连续解旋，包含数万次催化步骤，每个步骤都比改变动力学行为所需的构象变化时间快得多。这种配体依赖性的速率决定构象亚态的稳定导致 RecBCD 解旋酶活性在分子之间出现看似静态的变化，但实际上反映了单个分子在单个连续易位事件中表现出的平衡组合中的一个微态。
