Photocatalytic Proximity Labelling of MCL-1 by a BH3 Ligand

Abstract

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 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 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 manner with a variety of functional tags, as determined by in-gel fluorescence, affinity purification, and ESIMS analysis. In contrast with previous reports on Ru(II)(bpy)3-catalysed photolabelling, tandem MS experiments revealed that the dominant labelling 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 methodology for proximity-induced photolabelling of 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 and interactome of BCL-2 family proteins.<br>

Photocatalysis in Chemical Biology: Extending the Scope of Optochemical Control and Towards New Frontiers in Semisynthetic Bioconjugates and Biocatalysis

“…Optimization of this chemistry led to the identification of 1‐methyl‐4‐aryl‐urazole (MAUra, Figure 2 ,C ) as an excellent reagent in this labeling chemistry [19] . More recent work with a ligand‐Ru(bpy) 3 conjugate targeting MCL‐1, an important target in the apoptosis pathway, achieved selective labeling of its target with a variety of functional tags, with analysis showing that in this case, conjugation proceeded at a cysteine residue [20] . The selectivity for cysteine residue over a tyrosine residue is presumed to be the product of steric predisposition.…”

“…[19] More recent work with a ligand-Ru(bpy) 3 conjugate target- ing MCL-1, an important target in the apoptosis pathway, achieved selective labeling of its target with a variety of functional tags, with analysis showing that in this case, conjugation proceeded at a cysteine residue. [20] The selectivity for cysteine residue over a tyrosine residue is presumed to be the product of steric predisposition. Yet another example of proximity labeling was used to label lectins by employing beads 2,C).…”