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Beard et al ChemRxiv 2019.pdf (3.47 MB)

Photocatalytic Proximity Labelling of MCL-1 by a BH3 Ligand

preprint
submitted on 19.03.2019, 10:13 and posted on 19.03.2019, 19:33 by Hester Beard, Rachel George, Andrew Wilson, Robin Bon
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.

Funding

EPSRC: EP/N013573

EPSRC: EP/KO39292/1

Wellcome Trust: 097827/Z/11/A

Wellcome Trust: WT094232MA

Wellcome Trust: 094232/Z/10/Z

BBSRC: BB/M012573/1

History

Email Address of Submitting Author

r.bon@leeds.ac.uk

Institution

University of Leeds

Country

United Kingdom

ORCID For Submitting Author

0000-0003-1733-3680

Declaration of Conflict of Interest

None

Version Notes

ChemRxiv version 1

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