title: Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli.
abstract: F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan.
authors.forename: Ikuko
authors.initials: I
authors.lastname: Yumen
authors.forename: Iku
authors.initials: I
authors.lastname: Iwasaki
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Suzuki
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Todokoro
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tanaka
authors.forename: Osamu
authors.initials: O
authors.lastname: Okada
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
authors.forename: Hideo
authors.initials: H
authors.lastname: Akutsu
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003470:Culture Media; D046508:Culture Techniques; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D057075:Enzyme Assays; D004926:Escherichia coli; D008051:Lipid Bilayers / Q000737:chemistry*; D008567:Membranes, Artificial; D025261:Mitochondrial Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D011994:Recombinant Proteins; C103607:mitochondrial ATPase subunit c; D025261:Mitochondrial Proton-Translocating ATPases; D004134:Dimyristoylphosphatidylcholine