Substitution of the cysteinyl residue (Cys21) of subunit <i>b</i> of the ATP synthase from <i>Escherichia coli</i>

Kauffer, S.; Deckers-Hebestreit, Gabriele; Altendorf, Karlheinz

doi:10.1111/j.1432-1033.1991.tb16504.x

Cited by 5 publications

(1 citation statement)

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“…Conditions-Plasmid pTOM3.1 was constructed by cloning a 144-bp EcoNI fragment from pSK1 (28) as well as a 478-bp PpuMI/BssHI fragment from pRR76 (29) into plasmid pBWU13 (atpIЈBEFHAGDC) (30), thereby introducing the substitutions bC21A and bQ64C. Addition of a polyhistidine motif following the N-terminal methionine residue of subunit a was achieved by the site-directed introduction of a (CATCAC) 6 sequence via a two-stage PCR mutagenesis procedure (31), yielding plasmid pTOM3.1aHis 12 .…”

Section: Construction Of Plasmids and Growthmentioning

confidence: 99%

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

Krebstakies

Zimmermann

Gräber

et al. 2005

Journal of Biological Chemistry

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In F 1 F 0 -ATP synthase, the subunit b 2 ␦ complex comprises the peripheral stator bound to subunit a in F 0 and to the ␣ 3 ␤ 3 hexamer of F 1 . During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F 0 subunits to the binding of F 1 within the assembled holoenzyme, the isolated subunit b dimer, ab 2 subcomplex, and fully assembled F 0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys 64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F 1 (at ␥Cys 106 ) and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F 1 dissociation constants of 2.7 nM for the binding of F 0 and 9 -10 nM for both the ab 2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F 0 contribute to F 1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F 1 to F 0 , whereas subunit a does not contribute significantly.

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Section: Construction Of Plasmids and Growthmentioning

confidence: 99%

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

Krebstakies

Zimmermann

Gräber

et al. 2005

Journal of Biological Chemistry

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The ATP synthase of Escherichia coli: structure and function of F0 subunits

Deckers-Hebestreit

Greie

Stalz

et al. 2000

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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In this review we discuss recent work from our laboratory concerning the structure and/or function of the F(0) subunits of the proton-translocating ATP synthase of Escherichia coli. For the topology of subunit a a brief discussion gives (i) a detailed picture of the C-terminal two-thirds of the protein with four transmembrane helices and the C terminus exposed to the cytoplasm and (ii) an evaluation of the controversial results obtained for the localization of the N-terminal region of subunit a including its consequences on the number of transmembrane helices. The structure of membrane-bound subunit b has been determined by circular dichroism spectroscopy to be at least 75% alpha-helical. For this purpose a method was developed, which allows the determination of the structure composition of membrane proteins in proteoliposomes. Subunit b was purified to homogeneity by preparative SDS gel electrophoresis, precipitated with acetone, and redissolved in cholate-containing buffer, thereby retaining its native conformation as shown by functional coreconstitution with an ac subcomplex. Monoclonal antibodies, which have their epitopes located within the hydrophilic loop region of subunit c, and the F(1) part are bound simultaneously to the F(0) complex without an effect on the function of F(0), indicating that not all c subunits are involved in F(1) interaction. Consequences on the coupling mechanism between ATP synthesis/hydrolysis and proton translocation are discussed.

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THE F₀F₁-TYPE ATP SYNTHASES OF BACTERIA: Structure and Function of the F₀ Complex

Deckers-Hebestreit

Altendorf

1996

Annu. Rev. Microbiol.

190

162

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Membrane-bound ATP synthases (F0F1-ATPases) of bacteria serve two important physiological functions. The enzyme catalyzes the synthesis of ATP from ADP and inorganic phosphate utilizing the energy of an electrochemical ion gradient. On the other hand, under conditions of low driving force, ATP synthases function as ATPases, thereby generating a transmembrane ion gradient at the expense of ATP hydrolysis. The enzyme complex consists of two structurally and functionally distinct parts: the membrane-integrated ion-translocating F0 complex and the peripheral F1 complex, which carries the catalytic sites for ATP synthesis and hydrolysis. The ATP synthase of Escherichia coli, which has been the most intensively studied one, is composed of eight different subunits, five of which belong to F1, subunits alpha, beta, gamma, delta, and epsilon (3:3:1:1:1), and three to F0, subunits a, b, and c (1:2:10 +/- 1). The similar overall structure and the high amino acid sequence homology indicate that the mechanism of ion translocation and catalysis and their mode of coupling is the same in all organisms.

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Substitution of the cysteinyl residue (Cys21) of subunit b of the ATP synthase from Escherichia coli

Cited by 5 publications

References 53 publications

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

The ATP synthase of Escherichia coli: structure and function of F0 subunits

THE F₀F₁-TYPE ATP SYNTHASES OF BACTERIA: Structure and Function of the F₀ Complex

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Substitution of the cysteinyl residue (Cys21) of subunit b of the ATP synthase from Escherichia coli

Cited by 5 publications

References 53 publications

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

The ATP synthase of Escherichia coli: structure and function of F0 subunits

THE F0F1-TYPE ATP SYNTHASES OF BACTERIA: Structure and Function of the F0 Complex

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THE F₀F₁-TYPE ATP SYNTHASES OF BACTERIA: Structure and Function of the F₀ Complex