Chemical protein labelling for structural proteomics and interactome analyses

Informatics methods have established the protein-protein “interactome” as comprising from 130,000 to 650,000 complexes. Understanding the mechanistic role of proteins and their macromolecular complexes in signalling pathways requires the capability to study their interactome and localisation with temporal resolution. Similarly, although structural methods such as cryoEM, NMR and x-ray crystallography have been and continue to be enormously powerful in providing structural insight on the molecular mechanisms of functional biomacromolecular complexes, challenges remain in studying the transient interactions of intrinsically disordered regions of such complexes and whole interactomes in a cellular context. Our group are developing synthetic protein labelling chemistry that can provide solutions for these problems. Recently, our group introduced a suite of ‘tag and transfer’ diazirine-based cross-linking reagents; diazirines are ideal cross-linking groups because upon excitation with UV light, they generate highly reactive carbenes capable of indiscriminate insertion into proximal bonds. In effect, the reagents transfer a chemical label from one protein (the bait) onto another protein or proteins (the target). Proteolysis of the target protein followed my mass-spectrometry based sequencing then allows the location of the chemical label, thus revealing structural information on the nature of the interaction between bait and target. In a second, related approach we have developed photocatalytic proximity labelling methods whereby a ligand functionalized with a ruthenium (II) based photocatalyst is used to induce selective labelling of the ligands’ protein target through reaction with a third labelling reagent. Ongoing development of these methods and application to cellular systems could support mechanistic understanding of target biology earlier in the drug discovery process and reduce attrition in the more costly stages of clinical drug testing and development . 

 

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Key Papers

H. A. Beard, J. R. Hauser, M. Walko, R. M. George, A. J.Wilson, R. S. Bon: Photocatalytic proximity labelling of MCL-1 by a BH3 ligand, Comms. Chem., 2019, 2, 133. View Paper.

J. E. Horne, M. Walko, A. N. Calabrese, M. A. Levenstein, D. J. Brockwell, N. Kapur, A. J. Wilson, S. E. Radford FRS: RapidMapping of Protein Interactions Using Tag-TransferPhotocrosslinkers, Angew. Chemie. Int. Ed., 2018, 57, 16688–16692. View Paper.

G.W. Preston, S. E. Radford, A. E. Ashcroft, A. J. Wilson: Analysis of Amyloid Nanostructures Using Photo-Crosslinking: In Situ Comparison of Three Widely Used Photo-Crosslinkers, ACS Chem. Biol., 2014, 9, 761–768. View Paper.

G.W. Preston, S. E. Radford, A. E. Ashcroft, A. J. Wilson: Covalentcross-linking within supramolecular peptide structures, Anal. Chem., 2012, 84, 6790–6797. View Paper.

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Collaborators

Robin Bon (University of Leeds) and Sheena Radford (University of Leeds) 

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Current and Recent Funding

Royal Society Leverhulme Trust Senior Fellowship SRF\R1\191087: ‘In cell photo-labelling – A New Toolkit for Selective Tagging of Proteins for Dynamic Interactome Analysis’  

EPSRC EP/N035267/1 :‘Designer Chemistry to Probe Supramolecular Assembly Mechanism and Function’   

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Related Areas  

Understanding amyloid assembly mechanisms