title: Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.
abstract: The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.
authors.affiliation: The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Edgar
authors.initials: E
authors.lastname: Morales-Rios
authors.affiliation: The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Ian N
authors.initials: IN
authors.lastname: Watt
authors.affiliation: The Babraham Institute, Cambridge CB22 3AT, UK.
authors.forename: Qifeng
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Shujing
authors.initials: S
authors.lastname: Ding
authors.affiliation: The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Fearnley
authors.affiliation: The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Montgomery
authors.affiliation: The Babraham Institute, Cambridge CB22 3AT, UK.
authors.forename: Michael J O
authors.initials: MJ
authors.lastname: Wakelam
authors.affiliation: The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK walker@mrc-mbu.cam.ac.uk.
authors.forename: John E
authors.initials: JE
authors.lastname: Walker
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011485:Protein Binding; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases
keywords: F-ATPase; Paracoccus denitrificans; cardiolipin; crystallization; subunits; α-proteobacteria