- 作者：Chunli Zhang ; Matteo Allegretti ; Janet Vonck ; Julian D. Langer ; Marco Marcia ; Guohong Peng ; Hartmut Michel
- 关键词：IPTG ; isopropyl-尾-thiogalactopyranoside ; DM ; n-decyl-尾-d-maltoside ; DDM ; n-dodecyl-尾-d-maltoside ; 伪-PCC ; trans-4-(trans-4&prime ; -propylcyclohexyl)cyclohexyl-伪-d-maltoside ; BN-PAGE ; blue native polyacrylamide gel electrophoresis ; IMAC ; immobilized-metal affinity chromatography ; Ni-NTA ; Ni&ndash ; Nitrilotriacetic acid ; SEC ; size-exclusion chromatography ; PMF ; peptide mass fingerprint ; EM ; electron microscopy ; LC&ndash ; ESI&ndash ; MS ; liquid chromatography&ndash ; electrospray ionization tandem mas
- 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
- 出版年：January, 2014
- 年：2014
- 卷：1840
- 期：1
- 页码：34-40
- 全文大小：698 K
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.
General significance
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.