title: Tandem mass spectrometry defines the stoichiometry and quaternary structural arrangement of tryptophan molecules in the multiprotein complex TRAP.
abstract: We have used tandem mass spectrometry to examine the stoichiometry and binding sites of trp molecules in various assemblies of the protein complex TRAP. The results show that TRAP forms oligomers containing 11 and 12 subunits. MS/MS experiments show that up to 11 trp molecules bind to the 12-mer but that during gas-phase dissociation 5 then 6 trp molecules are released reflecting the different gas-phase stabilities of the partially ligated forms. At high trp concentrations, the protein assembles to form a double ring structure. Tandem mass spectrometry reveals that it is composed of 24 subunits with up to 22 molecules of trp. Dissociation of the complex reveals the same dissociation pathway as for the single ring structure, allowing us to propose a model for the assembly of the TRAP 24-mer based on the different environments of trp molecules. More generally, these results demonstrate the power of tandem mass spectrometry for defining the stoichiometry and quaternary structural arrangement of subunits and ligands within a 46-component multiprotein multiligand complex.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Margaret G
authors.initials: MG
authors.lastname: McCammon
authors.forename: Helena
authors.initials: H
authors.lastname: Hernández
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002021:Buffers; D018360:Crystallography, X-Ray; D013058:Mass Spectrometry; D016601:RNA-Binding Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014157:Transcription Factors / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002021:Buffers; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D014364:Tryptophan