title: Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase.
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.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: Joachim
authors.initials: J
authors.lastname: Weber
authors.forename: Susan
authors.initials: S
authors.lastname: Wilke-Mounts
authors.forename: Sashi
authors.initials: S
authors.lastname: Nadanaciva
authors.forename: Alan E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004492:Edetic Acid; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases