A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes.
A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes.
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DOI：
10.1371/journal.pone.0280693
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发表时间：
2023
期刊：
影响因子：
3.7
通讯作者：
Robertson, Janice
中科院分区：
文献类型：
--
作者：
Ernst, Melanie;Ozturk, Tugba;Robertson, Janice
膜蛋白经常被观察到作为高阶低聚物，在某些情况下，在多个化学计量的形式，提出了动态寡聚化是否可以连接到功能的调制的问题。为了更好地理解这种潜在的调节机制，有一个正在进行的努力，以量化直接在膜中的膜蛋白寡聚化的平衡反应。单分子光漂白分析对此特别有用，因为它提供了在稀释条件下附着于蛋白质亚基的荧光团的二进制读数。然而，化学计量的任何定量还关键地需要知道亚基被荧光标记的概率。由于标记的不确定性往往是不可避免的，我们开发了一种方法来估计标记产量使用的光漂白概率分布的内在二聚体控制。通过迭代拟合的实验二聚体的光漂白概率分布的预期二聚体模型，我们估计的荧光团标记的产量，并找到协议与直接测量标记的纯化蛋白质的UV-VIS吸收重建前。使用这种标记预测，类似的估计方法被应用于确定反应性CLC-ec 1二聚化构建体的解离常数，而无需荧光团标记产率的先验知识。最后，我们估计的操作范围的亚基标记产量，允许在整个反应范围内的摩尔分数密度的单体和二聚体群体的歧视。因此，我们的研究绘制了一个实用的方法，直接从单分子光漂白数据定量荧光标记，提高了定量反应性膜蛋白在膜中的化学计量的能力。
Membrane proteins are often observed as higher-order oligomers, and in some cases in multiple stoichiometric forms, raising the question of whether dynamic oligomerization can be linked to modulation of function. To better understand this potential regulatory mechanism, there is an ongoing effort to quantify equilibrium reactions of membrane protein oligomerization directly in membranes. Single-molecule photobleaching analysis is particularly useful for this as it provides a binary readout of fluorophores attached to protein subunits at dilute conditions. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Since labeling uncertainty is often unavoidable, we developed an approach to estimate labeling yields using the photobleaching probability distribution of an intrinsic dimeric control. By iterative fitting of an experimental dimeric photobleaching probability distribution to an expected dimer model, we estimate the fluorophore labeling yields and find agreement with direct measurements of labeling of the purified protein by UV-VIS absorbance before reconstitution. Using this labeling prediction, similar estimation methods are applied to determine the dissociation constant of reactive CLC-ec1 dimerization constructs without prior knowledge of the fluorophore labeling yield. Finally, we estimate the operational range of subunit labeling yields that allows for discrimination of monomer and dimer populations across the reactive range of mole fraction densities. Thus, our study maps out a practical method for quantifying fluorophore labeling directly from single-molecule photobleaching data, improving the ability to quantify reactive membrane protein stoichiometry in membranes.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
通过单分子荧光显微镜测量脂质双层中的膜蛋白二聚化平衡。
影响因子：
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作者：
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通讯作者：
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作者：
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影响因子：
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作者：
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通讯作者：
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作者：
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Patapoutian, Ardem