Conformational transitions of proteins are governed by chemical kinetics, often toggled by passage through an activated state separating two conformational ensembles. The passage time of a protein through the activated state can be too fast to be detected by single-molecule experiments without the aid of viscogenic agents. Here, we use high-bandwidth nanopore measurements to resolve microsecond-duration transitions that occur between conformational states of individual protein molecules partly blocking pore current. We measure the transition state passage time between folded and unfolded states of a two-state λ_6–85 mutant and between metastable intermediates and the unfolded state of the multistate folder cytochrome c. Consistent with the principle of microscopic reversibility, the transition state passage time is the same for the forward and backward reactions. A passage time distribution whose tail is broader than a single exponential observed in cytochrome c suggests a multidimensional energy landscape for this protein.

中文翻译：

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通过纳米孔离子电流跳跃直接观察单蛋白过渡态通过

蛋白质的构象转变受化学动力学控制，通常通过通过分离两个构象集合的激活状态来切换。蛋白质通过活化状态的通过时间可能太快而无法通过单分子实验检测到，而无需使用粘胶剂。在这里，我们使用高带宽纳米孔测量来解决在部分阻断孔电流的单个蛋白质分子的构象状态之间发生的微秒级跃迁。我们测量了两态 λ _6-85突变体的折叠和展开状态之间以及亚稳态中间体和多态折叠细胞色素c的展开状态之间的过渡态通过时间. 与微观可逆性原理一致，正向和反向反应的过渡态通过时间相同。其尾部比在细胞色素c中观察到的单个指数更宽的通道时间分布表明该蛋白质具有多维能量景观。