Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).
Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).
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DOI：
10.1038/srep28186
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发表时间：
2016-06-24
影响因子：
4.6
通讯作者：
French PM
中科院分区：
文献类型：
--
作者：
Margineanu A;Chan JJ;Kelly DJ;Warren SC;Flatters D;Kumar S;Katan M;Dunsby CW;French PM
我们提出了一种基于多孔板细胞的高含量分析方法，利用自动荧光寿命成像(FLIM)读出的Förster共振能量转移(FRET)来直接定量细胞中的蛋白质相互作用。自动拍摄采用广域时间门控检测，通常每个视场(FOV)只需要10 S。FOV的100‘S至1000’S对生物、热和散粒噪声进行平均，使具有高统计能力的无偏定量分析成为可能。绘制平均施主寿命与受体/施主强度比的图清楚地识别了蛋白质的相互作用，并通过对双指数施主衰变模型的拟合，估计了相互作用的布居比例，通过校准施主和受体的荧光强度，可以得到解离常数。我们展示了它在识别MST1蛋白结合伙伴和估计RASSF蛋白家族成员之间的相互作用强度方面的应用，RASSF蛋白家族通过Hippo信号通路在细胞凋亡中发挥重要作用。Kd值与公布的生化测量结果大体一致。
We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.