Beard Hester A, Hauser Jacob R, Walko Martin, George Rachel M, Wilson Andrew J, Bon Robin S
School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK.
Commun Chem. 2019 Nov 21;2:133. doi: 10.1038/s42004-019-0235-z.
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))-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))-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)-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. 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能够在无需基因编码标签的情况下，将多种化学官能团引入蛋白质中。在此，我们报告了一种使用钌联吡啶（Ru(II)(bpy)）修饰的肽对蛋白质进行快速标记的方法，该肽被设计用于模拟BCL-2家族蛋白质-蛋白质相互作用中的相互作用BH3配体。使用亚化学计量量的（Ru(II)(bpy)）修饰的NOXA-B并在可见光下照射1分钟，抗凋亡蛋白MCL-1可以用多种功能标签进行光标记。与先前关于Ru(II)(bpy)介导的光标记的报道不同，串联质谱实验表明标记位点是MCL-1的一个半胱氨酸残基。MCL-1可以在与其他蛋白质（包括结构相关的BCL-2成员BCL-x）的混合物中被选择性标记。这些结果表明，邻近诱导的光标记适用于介导蛋白质-蛋白质相互作用的界面，并为未来使用配体导向的邻近标记对BCL-2家族蛋白质的相互作用组进行动态分析铺平了道路。
