title: Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding.
abstract: Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1(Ubix), the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.
authors.affiliation: Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Beveridge
authors.affiliation: Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Lukasz G
authors.initials: LG
authors.lastname: Migas
authors.affiliation: Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Karl A P
authors.initials: KAP
authors.lastname: Payne
authors.affiliation: Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
authors.affiliation: Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.affiliation: Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Perdita E
authors.initials: PE
authors.lastname: Barran
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation
chemical_list: D003067:Coenzymes; D029968:Escherichia coli Proteins; D005486:Flavin Mononucleotide; D002262:Carboxy-Lyases; C113304:ubiX protein, E coli