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Photocatalytic proximity labelling of MCL-1 by a BH3 ligand
Communications Chemistry ( IF 5.9 ) Pub Date : 2019-11-21 , DOI: 10.1038/s42004-019-0235-z
Hester A. Beard , Jacob R. Hauser , Martin Walko , Rachel M. George , Andrew J. Wilson , Robin S. Bon

Ligand-directed protein labelling allows the introduction of diverse chemical functionalities onto proteins without the need for genetically encoded tags. Here we report a method for the rapid labelling of a protein using a ruthenium-bipyridyl (Ru(II)(bpy)3)-modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interactions. Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 min, the anti-apoptotic protein MCL-1 can be photolabelled with a variety of functional tags. In contrast with previous reports on Ru(II)(bpy)3-mediated photolabelling, tandem mass spectrometry experiments reveal that the labelling site is a cysteine residue of MCL-1. MCL-1 can be labelled selectively in mixtures with other proteins, including the structurally related BCL-2 member, BCL-xL. These results demonstrate that proximity-induced photolabelling is applicable to interfaces that mediate protein-protein interactions, and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the interactome of BCL-2 family proteins.



中文翻译:

BH3配体对MCL-1的光催化接近标记

配体导向的蛋白质标记可将多种化学功能引入蛋白质,而无需遗传编码的标签。在这里,我们报告了一种使用钌-联吡啶(Ru(II)(bpy)3)修饰的肽快速标记蛋白质的方法,该肽旨在模拟BCL-2家族蛋白质-蛋白质相互作用中相互作用的BH3配体。使用亚化学计量的(Ru(II)(bpy)3)修饰的NOXA-B并用可见光照射1分钟,可以用各种功能标签对抗凋亡蛋白MCL-1进行光标记。与先前有关Ru(II)(bpy)3的报告相反介导的光标记,串联质谱实验揭示该标记位点是MCL-1的半胱氨酸残基。MCL-1可以与其他蛋白质(包括结构相关的BCL-2成员BCL-x L)混合选择性标记。这些结果表明,邻近诱导的光标记法适用于介导蛋白质-蛋白质相互作用的界面,并为将来使用配体定向邻近标记法动态分析BCL-2家族蛋白质的相互作用铺平了道路。

更新日期:2019-11-21
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