Quantitative time domain analysis of lifetime-based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions.
Quantitative time domain analysis of lifetime-based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions.
复制标题
使用荧光蛋白进行基于寿命的福斯特共振能量转移测量的定量时域分析：静态随机各向同性荧光团方向分布。
DOI：
10.1002/jbio.201700366
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发表时间：
2018
影响因子：
2.8
通讯作者：
Alexandrov Y
中科院分区：
文献类型：
--
作者：
Alexandrov Y
Förster共振能量转移（FRET）测量被广泛用于通过FRET效率对供体和受体荧光团的接近程度的依赖来获得关于分子相互作用和构象的信息。荧光寿命测量可以提供FRET效率和相互作用群体分数的定量分析。许多FRET实验利用高度特异性标记基因表达的荧光蛋白，适用于活细胞和生物体。不幸的是，在基于荧光寿命的FRET读数分析中，荧光团方向快速随机化的典型假设对于荧光蛋白是无效的，因为与其上态寿命相比，它们的旋转迁移率很慢。在这里，以前的分析有效的静态各向同性分布的荧光偶极子FRET测量被纳入新的软件，用于拟合供体发射衰减曲线。计算的FRET参数，包括摩尔人口分数，进行比较分析的模拟和实验的FRET数据的假设下，静态和动态的荧光团和中间制度之间的完全动态和静态的荧光团，和FRET对内的混合物，探索。最后，提出了一种方法来校正从拟合的发射从静态FRET对各向同性的角分布（不正确的）通常假设的动态FRET衰减模型的伪影。
Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime‐based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.