Proposal for simultaneous analysis of fluorescence intensity fluctuations and resonance energy transfer (IFRET) measurements
Proposal for simultaneous analysis of fluorescence intensity fluctuations and resonance energy transfer (IFRET) measurements
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DOI：
10.1088/2050-6120/ab9b68
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发表时间：
2020-06
影响因子：
3.2
通讯作者：
M. Stoneman;G. Biener;V. Raicu
中科院分区：
文献类型：
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作者：
M. Stoneman;G. Biener;V. Raicu
共振能量转移（RET）和荧光波动光谱（FFS）是用于定量活细胞中寡聚复合物内膜受体的自缔合的基于荧光的强大技术。然而，RET光谱法提取低聚物详细四级结构信息的能力有时取决于关于不同大小的低聚物的相对丰度的假设，而FFS技术可能提供低聚物大小信息，但不能提供四级结构细节，因为它们缺乏分子间距离的探针。在这份报告中，我们介绍了一种方法，我们称之为“强度波动和共振能量转移”（IFRET），它结合了分析施主和受主的强度波动与RET效率的测定。因为三个测量的量各自具有对受体摩尔分数（XA）的唯一依赖性，所以所有三个的同时全局拟合显著地减少了数据拟合和最适当的拟合模型的选择中的模糊性。我们证明了该方法的有效性模拟亮度和RET效率的数据，将单体，二聚体和四聚体的混合物，并表明IFRET分析提供了一个重大的改进，在确定正确的四级结构模型和提取的单体，二聚体和四聚体的相对丰度。可以想象的是，IFRET的分辨率提高可能提供深入了解受体寡聚化在存在和不存在同源配体的情况下的功能意义。
Resonance energy transfer (RET) and fluorescence fluctuation spectroscopies (FFS) are powerful fluorescence-based techniques for quantifying the self-association of membrane receptors within oligomeric complexes in living cells. However, RET spectrometry’s ability to extract information on the detailed quaternary structure of oligomers sometimes rests on assumptions regarding the relative abundances of oligomers of different sizes, while FFS techniques may provide oligomer size information but not quaternary structure details, as they lack a probe for inter-molecular distances. In this report, we introduce a method which we termed ‘intensity fluctuations and resonance energy transfer’ (IFRET), which combines analysis of donor and acceptor intensity fluctuations with RET efficiency determination. Because the three measured quantities each have a unique dependence on the acceptor mole fraction (XA), simultaneous global fitting of all three dramatically reduces ambiguity in the data fitting and choice of the most appropriate fitting model. We demonstrate the effectiveness of the method on simulated brightness and RET efficiency data incorporating mixtures of monomers, dimers, and tetramers and show that IFRET analysis provides a major improvement in both identifying the correct quaternary structure model and extracting the relative abundances of the monomers, dimers, and tetramers. Conceivably, the enhanced resolution of IFRET could potentially provide insight into the functional significance of receptor oligomerization in the presence and absence of cognate ligands.