Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

• Published in: Biochimica et biophysica acta Vol. 1840; no. 1; pp. 34 - 40
• Main Authors: Zhang, Chunli, Allegretti, Matteo, Vonck, Janet, Langer, Julian D., Marcia, Marco, Peng, Guohong, Michel, Hartmut
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2014
• Subjects: AAFF(O); adenosine diphosphate; adenosine monophosphate; adenosine triphosphate; Adenosine Triphosphate - metabolism; Animals; Aquifex; Artificial operon; bacteria; Blotting, Western; blue native polyacrylamide gel electrophoresis; BN-PAGE; Catalysis; Chromatography, Gel; DDM; EAFF(O); electron microscopy; enzyme activity; Escherichia coli; Escherichia coli - enzymology; gel chromatography; gel electrophoresis; gene expression; Gram-Negative Bacteria - enzymology; H+/K+-exchanging ATPase; H-transporting ATP synthase; Heterologous expression; Hydrolysis; Hyperthermophilic organism; IMAC; immobilized-metal affinity chromatography; Immunoglobulin G - immunology; IPTG; isopropyl-β-thiogalactopyranoside; LC–ESI–MS; liquid chromatography–electrospray ionization tandem mass spectrometry; MALDI-TOF-MS; mass spectrometry; matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; Membrane protein complex; Mitochondrial Proton-Translocating ATPases - genetics; Mitochondrial Proton-Translocating ATPases - immunology; Mitochondrial Proton-Translocating ATPases - metabolism; n-decyl-β-d-maltoside; n-dodecyl-β-d-maltoside; Ni-NTA; Ni–Nitrilotriacetic acid; nucleoporins; operon; Peptide Fragments - immunology; peptide mass fingerprint; phosphates; PMF; Protein assembly; Rabbits; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Respiratory enzyme; SEC; size-exclusion chromatography; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; stoichiometry; trans-4-(trans-4′-propylcyclohexyl)cyclohexyl-α-d-maltoside; Western blotting; α-PCC; Α. aeolicus ATP synthase heterologously produced in E. coli; Α. aeolicus ATP synthase isolated from native cells of A. aeolicus; MALDI-TOF-MS; EAF1FO; BN-PAGE; IMAC; Artificial operon; PMF; EM; DM; AAF1FO; DDM; α-PCC; SEC; Membrane protein complex; Protein assembly; IPTG; Respiratory enzyme; LC–ESI–MS; Ni-NTA; Hyperthermophilic organism; Heterologous expression; isopropyl-β-thiogalactopyranoside; size-exclusion chromatography; Ni–Nitrilotriacetic acid; trans-4-(trans-4′-propylcyclohexyl)cyclohexyl-α-d-maltoside; peptide mass fingerprint; immobilized-metal affinity chromatography; AAFF(O); electron microscopy; liquid chromatography–electrospray ionization tandem mass spectrometry; Α. aeolicus ATP synthase heterologously produced in E. coli; n-dodecyl-β-d-maltoside; Α. aeolicus ATP synthase isolated from native cells of A. aeolicus; EAFF(O); n-decyl-β-d-maltoside; matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; blue native polyacrylamide gel electrophoresis
• ISSN: 0304-4165; 0006-3002; 1878-2434; 1872-8006
• DOI: 10.1016/j.bbagen.2013.08.023

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. 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. 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. Our system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase. 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. [Display omitted] •A. aeolicus F1FO ATP synthase was produced in E. coli using an artificial operon.•The heterologously produced enzyme complex is as active as the native one.•The structures of the heterologous and native complexes are identical.•Heterologous expression allows characterizing unique properties of ATP synthase.