Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins
Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins
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DOI：
10.1529/biophysj.105.071241
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发表时间：
2006-03-01
影响因子：
3.4
通讯作者：
Ellenberg, J
中科院分区：
文献类型：
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作者：
Beaudouin, JL;Mora-Bermúdez, F;Ellenberg, J
在生理条件下对蛋白质相互作用进行定量表征对于系统生物学至关重要。绿色荧光蛋白标记蛋白质的荧光光漂白/激活实验经常用于此目的，但缺乏从这类数据中提取物理化学参数的可靠分析方法。在此，我们实施了一个反应 - 扩散模型，以确定蛋白质相互作用和扩散对荧光重新分布的影响。该模型经过验证，并应用于在活细胞中通过光激活探测的五种染色质相互作用蛋白质。我们发现，非常短暂的相互作用对于染色质蛋白质来说是常见的。它们所观察到的移动性受可用于扩散的游离蛋白质数量的限制，而不是受结合蛋白质的短停留时间限制。因此，单个蛋白质在局部扫描染色质以寻找结合位点，而不是在随机的核位置重新结合之前进行全局扩散。通过考虑真实的细胞几何形状和结合位点的不均匀分布，我们的模型提供了一个分析荧光标记因子移动性的通用框架。此外，它定义了荧光扰动实验的实验局限性，并强调了需要互补的方法来测量活细胞中的瞬时生化相互作用。
Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells.