The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments
The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments
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DOI：
10.1016/j.bpj.2014.01.039
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发表时间：
2014-03-18
影响因子：
3.4
通讯作者：
Hristova, Kalina
中科院分区：
文献类型：
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作者：
King, Christopher;Sarabipour, Sarvenaz;Hristova, Kalina
福斯特共振能量转移（FRET）实验经常用于研究细胞膜中的膜蛋白之间的相互作用。然而，除了序列特异性相互作用的FRET之外，这些实验总是记录了由于邻近FRET的贡献，当供体和受体在类似于100埃的距离内偶然地彼此接近时发生邻近FRET。这种效应并不反映膜中的特定相互作用，并且经常不被重视，尽管其幅度可能很大。在这里，我们开发了一个计算描述的接近FRET，模拟的情况下，接近FRET时，荧光蛋白被用来标记单体，二聚体，三聚体和四聚体膜蛋白，以及存在于单体-二聚体平衡的膜蛋白。我们还进行了严格的实验测量这种效果，通过识别膜受体，不关联在哺乳动物膜。我们测量的FRET效率之间的黄色荧光蛋白和mCherry标记的版本，这些受体在质膜衍生的囊泡作为受体浓度的函数。最后，我们证明了我们的预测很好地描述了实验测量。这里提出的工作带来了额外的严格FRET为基础的膜蛋白相互作用的研究，并应在膜生物物理学研究的广泛用途。
Forster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of similar to 100 angstrom. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous experimental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.