title: Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli.
abstract: BACKGROUND
  F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.
  
  
  METHODS
  We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.
  
  
  RESULTS
  We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F1FO ATP synthases.
  
  
  CONCLUSIONS
  Our system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase.
  
  
  CONCLUSIONS
  More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.
authors.affiliation: Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Max-von-Laue-Str. 3, D-60438 Frankfurt am Main, Germany.
authors.forename: Chunli
authors.initials: C
authors.lastname: Zhang
authors.forename: Matteo
authors.initials: M
authors.lastname: Allegretti
authors.forename: Janet
authors.initials: J
authors.lastname: Vonck
authors.forename: Julian D
authors.initials: JD
authors.lastname: Langer
authors.forename: Marco
authors.initials: M
authors.lastname: Marcia
authors.forename: Guohong
authors.initials: G
authors.lastname: Peng
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000276:immunology; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007074:Immunoglobulin G; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases
keywords: AAF(1)F(O); Artificial operon; BN-PAGE; DDM; DM; EAF(1)F(O); EM; Heterologous expression; Hyperthermophilic organism; IMAC; IPTG; LC–ESI–MS; MALDI-TOF-MS; Membrane protein complex; Ni-NTA; Ni–Nitrilotriacetic acid; PMF; Protein assembly; Respiratory enzyme; SEC; blue native polyacrylamide gel electrophoresis; electron microscopy; immobilized-metal affinity chromatography; isopropyl-β-thiogalactopyranoside; liquid chromatography–electrospray ionization tandem mass spectrometry; matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; n-decyl-β-d-maltoside; n-dodecyl-β-d-maltoside; peptide mass fingerprint; size-exclusion chromatography; trans-4-(trans-4′-propylcyclohexyl)cyclohexyl-α-d-maltoside; Α. aeolicus ATP synthase heterologously produced in E. coli; Α. aeolicus ATP synthase isolated from native cells of A. aeolicus; α-PCC