Beard, H., et al. Photocatalytic proximity labelling of MCL-1 by a BH3 ligand. 10.26434/chemrxiv.782006.v1

Ligand-directed protein labelling can be used to introduce diverse chemical functionalities onto proteins without the need for incorporation of genetically encoded tags. Here we report a method for the rapid and efficient labelling of a proteinusing a ruthenium-bipyridyl (Ru(II)(bpy)3) modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interaction (PPI). Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 minute, the anti-apoptotic protein MCL-1 was photolabelled in a ligand-dependent mannerwith a variety of functional tags, as determined by in-gel fluorescence, affinity purification, and ESI-MS analysis. In contrast with previous reports on Ru(II)(bpy)3-catalysed photolabelling, tandem MS experiments revealed that the dominantlabelling occurred on a cysteine residue of MCL-1. Labelling of MCL-1 occurred selectively in mixtures with other proteins, including the structurally related BCL-2 member,BCL-xL. These results improve methodologyfor proximity-inducedphotolabellingof proteins, demonstrate the approach is applicable to interfaces that mediate PPIs,and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the localisation andinteractome of BCL-2 family proteins.