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Cerulean Venus and VenusY67C FRET Reference Standards

机译：蔚蓝金星和金星Y67C FRET参考标准

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摘要

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.
机译：福斯特的共振能量转移（FRET）可用于研究活细胞中的蛋白质-蛋白质相互作用。已经设计并实施了多种测量FRET的方法；但是，这些方法的准确性未知，这使得即使不是不可能，也很难解释FRET效率值。由于缺乏可用于验证FRET测量的具有已知FRET效率的标准，因此存在此问题。绿色荧光蛋白光谱变体的出现以及易于使用的细胞转染技术为解决该问题提供了一个简单的解决方案：开发具有已知FRET效率的基因构建体，可以高保真度复制并自由分布。在这项研究中，产生了供体和受体之间的分离距离逐渐增大的荧光蛋白构建体，并使用荧光寿命光谱，敏化受体发射和光谱成像来测量FRET效率。由于每种方法的结果吻合良好，因此可以以高精度和高精度确定每种构建体的FRET效率值，从而有理由将其用作标准。

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