Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli

Yumen, Ikuko; Iwasaki, Iku; Suzuki, Toshiharu; Todokoro, Yasuto; Tanaka, Kentaro; Okada, Osamu; Fujiwara, Toshimichi; Yoshida, Masasuke; Akutsu, Hideo

doi:10.1016/j.pep.2012.02.005

Cited by 10 publications

(20 citation statements)

References 21 publications

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“…Compared with wild type, the amount of the Atp9p ring at 30 min was estimated to be approximately 47,20, and 8% in the mss51, atp10, and cox6 mutant, respectively (Fig. 4B).…”

Section: Properties Of the Atp9p-cox6pmentioning

confidence: 96%

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

McStay

Tzagoloff

2014

Journal of Biological Chemistry

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Background:The Atp9p rotor is an assembly module of mitochondrial ATP synthase. Results: Newly translated Atp9p and Cox6p, a subunit of yeast cytochrome oxidase (COX), are present in large complexes. Conclusion: Atp9p-Cox6p complexes serve as a source of Atp9p for rotor formation. Significance: We propose that Cox6p enhances the efficiency of Atp9p ring formation and may also coordinate balanced expression of COX and ATP synthase.

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“…Compared with wild type, the amount of the Atp9p ring at 30 min was estimated to be approximately 47,20, and 8% in the mss51, atp10, and cox6 mutant, respectively (Fig. 4B).…”

Section: Properties Of the Atp9p-cox6pmentioning

confidence: 96%

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

McStay

Tzagoloff

2014

Journal of Biological Chemistry

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“…Two-dimensional (2D) 13 C homonuclear correlation spectra with the dipolar assisted rotational resonance (DARR) pulse sequence 15 were obtained under magic angle sample spinning (MAS). The MAS rate was 12.5 kHz unless otherwise specified.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…1,2 The isolated F 1 is water-soluble. The membrane-embedded F o domain comprises ab 2 c [8][9][10][11][12][13][14][15] subunits, and forms a channel for transportation of proton. ATP synthase is a rotary motor, in which the c-subunit ring functions as a rotor.…”

Section: Introductionmentioning

confidence: 99%

“…ATP synthase is a rotary motor, in which the c-subunit ring functions as a rotor. Downhill proton flow through F o causes rotation of the rotor (c-ring) and rotation of the F 1 -γε (rotor stalk; F o c [8][9][10][11][12][13][14][15] -F 1 γε). The rotation of γ induces conformational changes in the β-subunits, which results in ATP synthesis at the F 1 catalytic sites.…”

Section: Introductionmentioning

confidence: 99%

“…Although a standard method was reported 13 , we need sophisticated techniques to obtain enough amount of the protein. In this work, we have tried to develop a more general procedure of TF o c-ring preparation for NMR measurements.…”

mentioning

confidence: 99%

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Improved Purification of Thermophilic F_oF₁-ATP Synthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes

Bak¹,

Kang²,

Suzuki³

et al. 2013

Journal of the Korean Magnetic Resonance Society

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ATP synthase produces ATP, a major energy source for metabolic processes in organisms, from ADP and inorganic phosphate in cellular membranes. ATP synthase is known as a rotary motor, in which the c-subunit ring functions as a rotor. In this work, we have tried to develop a more general preparation procedure of thermophilic F o c-ring (TF o c-ring) for NMR measurements. The expression of TF o F 1 is easily affected by various experimental conditions such as temperature, shape and size of a flask, a volume of medium, and shaking rate of an incubator. Accordingly, we have tried to optimize the expression conditions of TF o F 1 . TF o c-rings were purified from TF o F 1 according to a reported method. We modified purification procedures to improve purity and yield of TF o c. On top of them, we found a new combination of detergents for the purification at anion-exchange column chromatography. To examine the effect of lipid environments on the structure, the TF o c-rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the TF o c-ring structures in these membranes with solid-state NMR.

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Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure

et al. 2017

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FF-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H translocation in membranes. The F c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state C NMR (ssNMR) signals of the F c-subunit ring of thermophilic Bacillus PS3 (TF c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C-C'C correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue C-C chemical shift correlations, 97% of C, 97% of C and 92% of C' signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.

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Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli

Cited by 10 publications

References 21 publications

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

Improved Purification of Thermophilic F_oF₁-ATP Synthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes

Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure

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Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli

Cited by 10 publications

References 21 publications

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

Improved Purification of Thermophilic FoF1-ATP Synthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes

Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure

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Improved Purification of Thermophilic F_oF₁-ATP Synthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes