Claton Liam E, Baker Chrissy, Martin Hayes, Dzyuba Sergei V, Zaman Khadiza, Prokai Laszlo, Stewart Mikaela D, Simanek Eric E
Department of Chemistry & Biochemistry, Texas Christian University, Fort Worth, TX 76129, USA.
Department of Biology, Texas Christian University, Fort Worth, TX 76129, USA.
Biomolecules. 2024 Dec 18;14(12):1625. doi: 10.3390/biom14121625.
The functionalization of protein sidechains with highly water-soluble chlorotriazines (or derivatives thereof) using nucleophilic aromatic substitution reactions has been commonly employed to install various functional groups, including poly(ethylene glycol) tags or fluorogenic labels. Here, a poorly soluble dichlorotriazine with an appended indole is shown to react with a construct containing the disordered domain of BRCA1. Subsequently, this construct can undergo proteolytic cleavage to remove the SUMO-tag: the -terminal poly(His) tag is still effective for purification. Steady-state fluorescence, circular dichroism spectroscopy, and isothermal titration calorimetry with the binding partner of BRCA1, PALB2, are used to characterize the indole-labeled BRCA1. Neither the reaction conditions nor the indole-tag appreciably alter the structure of the BRCA1. Mass spectrometry confirms that the target is modified once, although the location of modification cannot be determined by tandem mass spectrometry with collision-induced dissociation due to disadvantageous fragmentation patterns.
利用亲核芳香取代反应，通过具有高水溶性的氯代三嗪（或其衍生物）对蛋白质侧链进行功能化修饰，已被广泛用于引入各种功能基团，包括聚乙二醇标签或荧光标记。在此，展示了一种带有附加吲哚的难溶性二氯三嗪与含有BRCA1无序结构域的构建体发生反应。随后，该构建体可进行蛋白水解切割以去除SUMO标签：其N端的聚组氨酸标签仍可有效地用于纯化。使用稳态荧光、圆二色光谱以及与BRCA1的结合伴侣PALB2进行等温滴定量热法来表征吲哚标记的BRCA1。反应条件和吲哚标签均未明显改变BRCA1的结构。质谱证实目标被修饰了一次，尽管由于不利的碎片模式，无法通过碰撞诱导解离的串联质谱法确定修饰位置。
