Quantitative FRET Analysis With the E0GFP-mCherry Fluorescent Protein Pair
Quantitative FRET Analysis With the E0GFP-mCherry Fluorescent Protein Pair
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DOI：
10.1111/j.1751-1097.2008.00435.x
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发表时间：
2009-01-01
影响因子：
3.3
通讯作者：
Beltram, Fabio
中科院分区：
文献类型：
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作者：
Albertazzi, Lorenzo;Arosio, Daniele;Beltram, Fabio
荧光蛋白之间的荧光共振能量转移(FRET)是研究活细胞中蛋白质相互作用甚至蛋白质修饰的有力工具。在这里，我们分析了E(0)GFP-mCherry对，并表明它可以在体内和体外产生可重复性的能量转移效率的定量测定。报道了这两种蛋白质的光物理性质，显示出良好的光谱重叠(Forster半径R-0=51埃)，受体和供体通道之间的低串扰，发射光谱不受pH和卤离子浓度的影响。受体光漂白(APB)和单、双光子荧光寿命成像显微镜(FLIM)被用来定量确定FRET效率值。介绍了一种基于串联结构的FRET标准，该结构包括供体和受体，以及一个20个氨基酸长的可切割的多肽连接体。参考值通过连接子的酶裂解获得，并被用作APB和FLIM数据的基准。E(0)GFP-mCherry具有理想的FRET薄膜检测性能，在体外和体内均有较高的准确度。此外，最近引入的相量方法被证明即使在次优的实验条件下也能得到直接和准确的双光子FRET效率数据。这些结果与参考方法(体外和体内)的一致性表明，这种新的对可以用于非常有效的定量FRET成像。
Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein-protein interaction and even protein modifications in living cells. Here, we analyze the E(0)GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro. The photophysics of the two proteins is reported and shows good spectral overlap (Forster radius R-0 = 51 angstrom), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E(0)GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo. Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo) reveals that this new pair can be used for very effective quantitative FRET imaging.