PMID:14722065
| Citation |
Weber, J, Wilke-Mounts, S, Nadanaciva, S and Senior, AE (2004) Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase. J. Biol. Chem. 279:11253-8 |
|---|---|
| Abstract |
The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)). With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm. Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1). The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions. |
| Links |
PubMed Online version:10.1074/jbc.M312576200 |
| Keywords |
Base Sequence; DNA Primers; Edetic Acid/chemistry; Escherichia coli/enzymology; Mitochondrial Proton-Translocating ATPases/genetics; Mitochondrial Proton-Translocating ATPases/metabolism; Mutagenesis, Site-Directed; Spectrometry, Fluorescence; Substrate Specificity |
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