FRET or no FRET: A quantitative comparison
FRET or no FRET: A quantitative comparison
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DOI：
10.1016/s0006-3495(03)75126-1
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发表时间：
2003-06-01
影响因子：
3.4
通讯作者：
Danuser, G
中科院分区：
文献类型：
--
作者：
Berney, C;Danuser, G
荧光共振能量转移（FRET）是一种用于通过能量从受激供体转移到受体来测量用两种不同荧光团（供体和受体）标记的两个分子之间的相互作用的技术。在生物学应用中，这种技术已经变得流行，以定性地映射蛋白质-蛋白质相互作用，并且在生物物理项目中，它被用作单个供体和受体分子之间距离的定量测量。在文献中可以找到许多方法来量化和绘制FRET，但它们提供的措施往往难以解释。我们在这里提出了这些方法的定量比较，通过使用表面FRET系统与控制量的供体和受体荧光团和控制它们之间的距离。我们支持该系统与Monte Carlo模拟的FRET，它提供了参考值的FRET效率在各种实验条件下。我们验证了一组具有代表性的FRET效率和指数计算不同的方法与不同的实验设置。最后，我们测试了它们的灵敏度，并得出结论，准备在更复杂和更少的控制系统的FRET实验。
Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein interactions, and in biophysical projects it is used as a quantitative measure for distances between a single donor and acceptor molecule. Numerous approaches can be found in the literature to quantify and map FRET, but the measures they provide are often difficult to interpret. We propose here a quantitative comparison of these methods by using a surface FRET system with controlled amounts of donor and acceptor fluorophores and controlled distances between them. We support the system with a Monte Carlo simulation of FRET, which provides reference values for the FRET efficiency under various experimental conditions. We validate a representative set of FRET efficiencies and indices calculated from the different methods with different experimental settings. Finally, we test their sensitivity and draw conclusions for the preparation of FRET experiments in more complex and less-controlled systems.