Yeow Edwin K L, Clayton Andrew H A
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637616.
Biophys J. 2007 May 1;92(9):3098-104. doi: 10.1529/biophysj.106.099424.
Protein-protein interactions play a pivotal role in biological signaling networks. It is highly desirable to perform experiments that can directly assess the oligomerization state and degree of oligomerization of biological macromolecules in their native environment. Homo-FRET depends on the inverse sixth power of separation between interacting like fluorophores on the nanometer scale and is therefore sensitive to protein oligomerization. Homo-FRET is normally detected by steady-state or time-resolved fluorescence anisotropy measurements. Here we show by theory and simulation that an examination of the extent of homotransfer as measured by steady-state fluorescence anisotropy as a function of fluorophore labeling (or photodepletion) gives valuable information on the oligomerization state of self-associating proteins. We examine random distributions of monomers, dilute solutions of oligomers, and concentrated solutions of oligomers. The theory is applied to literature data on band 3 protein dimers in membranes, GPI-linked protein trimers in "rafts," and clustered GFP-tagged epidermal growth factor receptors in cell membranes to illustrate the general utility and applicability of our analytical approach.
蛋白质-蛋白质相互作用在生物信号网络中起着关键作用。非常希望能够进行实验，直接评估生物大分子在其天然环境中的寡聚化状态和寡聚化程度。同源荧光共振能量转移（Homo-FRET）取决于纳米尺度上相互作用的同类荧光团之间距离的六次方反比，因此对蛋白质寡聚化敏感。同源荧光共振能量转移通常通过稳态或时间分辨荧光各向异性测量来检测。在此，我们通过理论和模拟表明，通过稳态荧光各向异性测量的同源转移程度作为荧光团标记（或光耗尽）的函数进行检查，可以提供关于自缔合蛋白质寡聚化状态的有价值信息。我们研究了单体的随机分布、寡聚物的稀溶液和寡聚物的浓溶液。该理论应用于关于膜中带3蛋白二聚体、“筏”中糖基磷脂酰肌醇（GPI）连接蛋白三聚体以及细胞膜中聚集的绿色荧光蛋白（GFP）标记的表皮生长因子受体的文献数据，以说明我们分析方法的一般实用性和适用性。
