title: Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.
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.
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Lucio
authors.initials: L
authors.lastname: Manzi
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Barrow
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Queen's Medical Centre, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Scott
authors.affiliation: Queen's Medical Centre, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Wright
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: John E
authors.initials: JE
authors.lastname: Moses
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
mesh_terms: D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D008024:Ligands; D008697:Methane / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry; D009113:Muramidase / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D023302:Protein Footprinting / Q000379:methods*; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D005960:Glucosides; D008024:Ligands; D015335:Molecular Probes; D009844:Oligosaccharides; D011506:Proteins; D025801:Ubiquitin; C511150:penta-N-acetyl-chitopentaose; C030011:carbene; C421864:hen egg lysozyme; D009113:Muramidase; D008697:Methane