Department of Physics and Biological Physics Research Group, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
Chemphyschem. 2011 Feb 25;12(3):571-9. doi: 10.1002/cphc.201000834. Epub 2011 Jan 30.
Switchable FRET is the combination of single-molecule Förster resonance energy transfer (smFRET) with photoswitching, the reversible activation and deactivation of fluorophores by light. By photoswitching, multiple donor-acceptor fluorophore pairs can be probed sequentially, thus allowing observation of multiple distances within a single immobilized molecule. Control of the photoinduced switching rates permits adjustment of the temporal resolution of switchable FRET over a wide range of timescales, thereby facilitating application to various dynamical biological systems. We show that fast total internal reflection (TIRF) microscopy can achieve measurements of two FRET pairs with 10 ms temporal resolution within less than 2 s. The concept of switchable FRET is also compatible with confocal microscopy on immobilized molecules, providing better data quality at high temporal resolution. To identify states and extract their transitions from switchable FRET time traces, we also develop linked hidden Markov modeling (HMM) of both FRET and donor-acceptor stoichiometry. Linked HMM successfully identifies transient states in the two-dimensional FRET-stoichiometry space and reconstructs their connectivity network. Improved temporal resolution and novel data analysis make switchable FRET a valuable tool in molecular and structural biology.
可切换的荧光共振能量转移（FRET）是单分子Förster 共振能量转移（smFRET）与光致变色的结合，即通过光可逆地激活和失活荧光团。通过光致变色，可以依次探测多个供体-受体荧光团对，从而允许在单个固定分子内观察多个距离。光诱导开关速率的控制允许在广泛的时间尺度范围内调整可切换 FRET 的时间分辨率，从而有利于应用于各种动态生物系统。我们表明，快速全内反射（TIRF）显微镜可以在不到 2 秒的时间内以 10 ms 的时间分辨率测量两个 FRET 对。可切换 FRET 的概念也与固定分子上的共聚焦显微镜兼容，在高时间分辨率下提供更好的数据质量。为了从可切换 FRET 时间轨迹中识别状态并提取它们的转变，我们还开发了 FRET 和供体-受体计量比的关联隐马尔可夫模型（HMM）。关联 HMM 成功地在二维 FRET-计量比空间中识别瞬态状态，并重建它们的连接网络。提高的时间分辨率和新颖的数据分析使可切换 FRET 成为分子和结构生物学的有力工具。
