Detection of Structural Dynamics by FRET: A Photon Distribution and Fluorescence Lifetime Analysis of Systems with Multiple States
Detection of Structural Dynamics by FRET: A Photon Distribution and Fluorescence Lifetime Analysis of Systems with Multiple States
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DOI：
10.1021/jp102156t
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发表时间：
2010-06-17
影响因子：
3.3
通讯作者：
Seidel, Claus A. M.
中科院分区：
文献类型：
--
作者：
Kalinin, Stanislav;Valeri, Alessandro;Seidel, Claus A. M.
提出了共焦单分子荧光光谱中的两种互补方法，通过考虑模拟和实验数据的福斯特共振能量转移（FRET）测量来分析构象动力学。首先，通过对不同时间段的 FRET 峰形状进行全局分析，应用光子分布分析 (PDA) 的扩展来表征两个或多个状态之间的构象交换。 PDA 可以在存在 FRET 波动的情况下准确预测 FRET 效率直方图的形状，同时考虑散粒噪声和背景贡献。 Dynamic-PDA 在扩散时间 t(d)（通常为毫秒）的时间尺度上定量恢复相互转换状态的 FRET 效率和动力学弛豫时间，该方法的动态范围相对于 t(d) 约为 +/-1 个数量级。提出校正程序以考虑限制动态 PDA 准确性的因素，例如亮度变化、由于扩散而缩短的观察时间以及多分子事件的贡献。其次，提出了多参数荧光检测的分析程序，其中强度衍生的 FRET 效率与 FRET 猝灭的供体的荧光寿命相关。如果应用最大似然估计器来计算混合态的平均荧光寿命，则可以获得荧光加权平均寿命。因此，通过荧光寿命的特征变化来检测混合状态，该荧光寿命变得比具有相同强度衍生的 FRET 效率的单一物种预期的更长。针对静态和动态 FRET 的情况，提供了用于直接目视检查 FRET 效率与供体寿命的二维图的分析工具。最后将这些新技术与荧光相关光谱进行比较。
Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Forster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied to characterize conformational exchange between two or more states via global analysis of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quantitatively recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time t(d) (typically milliseconds), with a dynamic range of the method of about +/-1 order of magnitude with respect to t(d). Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimolecular events. Second, an analysis procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a maximum likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Analysis tools for direct visual inspection of two-dimensional diagrams of FRET efficiency versus donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.