Kim Jae-Yeol, Kim Cheolhee, Lee Nam Ki
Department of Physics, Pohang University of Science and Technology, Pohang 790-784, Korea.
1] Department of Physics, Pohang University of Science and Technology, Pohang 790-784, Korea [2] School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Korea.
Nat Commun. 2015 Apr 24;6:6992. doi: 10.1038/ncomms7992.
Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s(-1) between two conformers of a Holliday junction. The rapid diffusional migration of Deinococcus radiodurans single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of Escherichia coli SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution.
单分子荧光共振能量转移（smFRET）是解析非同步生物分子动力学的强大技术之一。然而，smFRET在观察动力学的时间分辨率方面存在局限性。在此，我们报告了一种通过将分子 tethering 到自由扩散的100纳米大小脂质体上以亚毫秒分辨率观察实时动力学的新方法。扩散分子的观察时间延长至100毫秒且具有亚毫秒分辨率，这使得能够使用隐马尔可夫模型从FRET时间轨迹直接分析过渡态。我们测量了霍利迪连接体两个构象之间高达1500 s⁻¹的过渡速率。通过FRET解析了耐辐射球菌单链DNA结合蛋白（SSB）在单链DNA上的快速扩散迁移，其速度比大肠杆菌SSB快一个数量级。我们的方法是一种以亚毫秒分辨率研究生物分子动力学和运动的强大方法。 （注：原文中“tethering”这个词在给定语境下不太明确准确意思，可能是“系留”之类的意思，此处按原样保留未准确翻译。）
