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-x_L. 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.

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。