Analysis of binding reactions by fluorescence recovery after photobleaching
Analysis of binding reactions by fluorescence recovery after photobleaching
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DOI：
10.1529/biophysj.103.026765
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发表时间：
2004-06-01
影响因子：
3.4
通讯作者：
McNally, JG
中科院分区：
文献类型：
--
作者：
Sprague, BL;Pego, RL;McNally, JG
光漂白后荧光恢复（FRAP）现在被广泛用于研究活细胞中的结合相互作用。尽管已经确定了控制FRAP的反应-扩散方程的各种理想解，但还没有全面的分析或系统的方法来作为从任意FRAP曲线中提取结合信息的指南。在这里，我们给出了单个或多个独立结合相互作用的FRAP反应扩散方程的完整解，然后将我们的解与各种理想情况联系起来。这产生了一种连贯的方法，从FRAP数据中提取结合信息，我们已经将其应用于细胞核中转录因子迁移的问题。我们发现，在细胞核内，糖皮质激素受体是短暂地结合到一个单一的状态，每个分子每秒平均结合65个位点。这种快速采样可能对寻找特定的启动子靶序列很重要。此外，我们表明，这种主要的结合状态不是核基质，因为一些研究表明。我们举例说明了我们的分析如何对FRAP拟合提供几个自一致性检查。我们还定义了可以从FRAP数据中估计的约束条件，表明扩散应该在许多FRAP恢复中发挥关键作用，并提供了测试其贡献的工具。总的来说，我们的方法建立了一个更一般的框架来评估扩散的作用，结合状态的数量，以及FRAP恢复背后的结合常数。
Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding interactions in live cells. Although various idealized solutions have been identified for the reaction-diffusion equations that govern FRAP, there has been no comprehensive analysis or systematic approach to serve as a guide for extracting binding information from an arbitrary FRAP curve. Here we present a complete solution to the FRAP reaction-diffusion equations for either single or multiple independent binding interactions, and then relate our solution to the various idealized cases. This yields a coherent approach to extract binding information from FRAP data which we have applied to the question of transcription factor mobility in the nucleus. We show that within the nucleus, the glucocorticoid receptor is transiently bound to a single state, with each molecule binding on average 65 sites per second. This rapid sampling is likely to be important in finding a specific promoter target sequence. Further we show that this predominant binding state is not the nuclear matrix, as some studies have suggested. We illustrate how our analysis provides several self-consistency checks on a FRAP fit. We also define constraints on what can be estimated from FRAP data, show that diffusion should play a key role in many FRAP recoveries, and provide tools to test its contribution. Overall our approach establishes a more general framework to assess the role of diffusion, the number of binding states, and the binding constants underlying a FRAP recovery.