title: Chemical cross-linking, mass spectrometry, and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii.
abstract: A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrical barrel-like structures of four-stacked homoheptameric rings of α- and β-type subunits organized in α(7)β(7) β(7)α(7) stoichiometry. The CPs of H. volcanii are formed from a single type of β subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α1(7)β(7)β(7)α1(7) and α2(7)β(7)β(7)α2(7) CPs from H. volcanii. Chemically cross-linked peptides of the H. volcanii CPs were analyzed by high-performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross-linking mass spectrometry, together with the available structural data of nonhalophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1-, α2-, and β-homoheptameric rings from several different possible structures from Protein Data Bank.
authors.affiliation: Department of Chemistry, School of Medicine, University of Belgrade, Belgrade, Serbia.
authors.forename: Ivanka
authors.initials: I
authors.lastname: Karadzic
authors.forename: Julie
authors.initials: J
authors.lastname: Maupin-Furlow
authors.forename: Matthew
authors.initials: M
authors.lastname: Humbard
authors.forename: Laurence
authors.initials: L
authors.lastname: Prunetti
authors.forename: Pragya
authors.initials: P
authors.lastname: Singh
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry*; D003198:Computer Simulation; D019622:Haloferax volcanii / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment
chemical_list: D019843:Archaeal Proteins; D046988:Proteasome Endopeptidase Complex