Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition
Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition
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DOI：
10.1016/j.bpj.2009.07.044
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发表时间：
2009-10-21
影响因子：
3.4
通讯作者：
Tramier, Marc
中科院分区：
文献类型：
--
作者：
Padilla-Parra, Sergi;Auduge, Nicolas;Tramier, Marc
基于荧光蛋白质的荧光共振能量转移(FRET)方法是定量研究活细胞中蛋白质-蛋白质相互作用的一种有效方法，特别是与荧光寿命成像显微镜(FLIM)相结合时。为了比较不同FRET对在FRET-FLIM实验中的性能，我们首先测试了与不同红色受体(mRFP1-EGFP、mStrawberry-EGFP、HaloTag(TMR)-EGFP和mCherry-EGFP)连接的增强型绿色荧光蛋白(EGFP)。我们使用假设双物种模型的不同数学模型(即，离散双指数固定施主寿命和双指数拉伸FRET寿命)，得到了参与FRET的一部分施主(f(D))，这与理想的一种情况相差很远。我们表明，用这些模型得到的相对较低的f(D)百分比可能是由于受主群体的光谱不均一性造成的，这部分是由于施主和受主的不同成熟率造成的。为了提高参与FRET的供体蛋白的数量，我们测试了mTFP1作为供体分别与Morange和EYFP偶联。在供体FRET-FILM实验中，mTFP1至少与EGFP一样好，因为1)它的寿命在光诱导的荧光变化过程中保持不变；2)它的荧光衰减曲线最好地符合单指数模型；3)没有检测到光转化。以EYFP为受试物时，f(D)值最高(0.7)。此外，在快速获取的背景下，我们获得了mTFP1-EYFP的最小f(D)(Mf(D))，几乎是mCherry-EGFP的两倍(0.65比0.35)。最后，我们在融合蛋白高度固定的生物学情况下比较了EGFP和mTFP1，并且在快速电影获取中，EGFP和mTFP1与组蛋白H4(EGFP-H4和mTFP1-H4)相连。在这种特殊情况下，EGFP-H4的荧光强度比mTFP1-H4更稳定。然而，我们发现，在f(D)分析中，mTFP1/EYFP单独是最好的FRET-Flim对。
The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f(D) value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f(D) (mf(D)) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f(D) analysis.