Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

Abstract

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Citation

Manzi, L., Barrow, A. S., Scott, D., Layfield, R., Wright, T. G., Moses, J. E., & Oldham, N. J. (2016). Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions. Nature Communications, 7, Article 13288. https://doi.org/10.1038/ncomms13288

• Journal Article Type: Article
• Acceptance Date: Sep 20, 2016
• Publication Date: Nov 16, 2016
• Deposit Date: Nov 1, 2016
• Publicly Available Date: Nov 23, 2016
• Journal: Nature Communications
• Electronic ISSN: 2041-1723
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 7
• Article Number: 13288
• DOI: https://doi.org/10.1038/ncomms13288
• Public URL: https://nottingham-repository.worktribe.com/output/828258
• Publisher URL: http://www.nature.com/articles/ncomms13288
• Contract Date: Nov 1, 2016