A fluorescence assay for leucine zipper dimerization: Avoiding unintended consequences of fluorophore attachment
A fluorescence assay for leucine zipper dimerization: Avoiding unintended consequences of fluorophore attachment
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DOI：
10.1021/ja990178p
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发表时间：
1999-05-12
影响因子：
15
通讯作者：
Gellman, SH
中科院分区：
文献类型：
--
作者：
Daugherty, DL;Gellman, SH
α-螺旋卷曲螺旋二聚体的形成代表了特定蛋白质-蛋白质相互作用的最简单的例子之一。这种二聚化模式在转录调节蛋白中常见，该过程被称为亮氨酸拉链形成。亮氨酸拉链二聚化的抑制剂将允许人们控制基因表达。作为识别此类抑制剂的第一步，我们开发了一种基于荧光的检测方法，用于检测与 Jun 蛋白的亮氨酸拉链区域相对应的肽的同二聚化。该测定涉及将 7-羟基香豆素附着到 Jun 肽的 N 末端。二聚化后，成对的荧光团彼此靠近，从而导致自猝灭。二聚体的破坏通过 7-羟基香豆素荧光的增加来表示。事实证明，该测定的开发比预期更复杂，因为我们发现两种常用的荧光标签，芘和荧光素，会诱导亮氨酸拉链-荧光团缀合物的高阶聚集。这种聚集被证明是荧光团和肽部分之间的协同作用的结果。第三种常见荧光团 7-二乙氨基香豆素可显着稳定 Jun 同型二聚体。在我们检查的四种荧光团中，7-羟基香豆素对亮氨酸拉链肽行为的干扰最小。
Formation of alpha-helical coiled-coil dimers represents one of the simplest examples of a specific protein-protein interaction. This dimerization mode is commonly observed among transcription regulator proteins, where the process is referred to as leucine zipper formation. Inhibitors of leucine zipper dimerization would allow one to control gene expression. As a first step toward identifying such inhibitors, we have developed a fluorescence-based assay for homodimerization of peptides corresponding to the leucine zipper region of the Jun protein. The assay involves attachment of 7-hydroxycoumarin to the N-terminus of the Jun peptide. Upon dimerization, pairs of fluorophores are held near one another, which leads to self-quenching. Disruption of the dimer is signaled by an increase of 7-hydroxycoumarin fluorescence. Development of this assay proved to be more complex than expected, because we found that two commonly used fluorescent tags, pyrene and fluorescein, induce high-order aggregation of leucine zipper-fluorophore conjugates. This aggregation was demonstrated to result from cooperativity between the fluorophore and peptide portions. A third common fluorophore, 7-diethylaminocoumarin, significantly stabilized the Jun homodimer. Of the four fluorophores we examined, 7-hydroxycoumarin caused the least perturbation of leucine zipper peptide behavior.