Quantitative Determination of Direct Binding of b Subunit to F1 in Escherichia coli F1F0-ATP Synthase

Joachim Weber, Susan Wilke-Mounts, Sashi Nadanaciva, Alan E. Senior

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24 Scopus citations


The stator in F1F0-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b2δ subunit complex comprises the stator, bound to subunit a in F0 and to the α3β3 hexagon of F1. To quantitatively characterize binding of b subunit to the F1 α3β3 hexagon, we developed fluorimetric assays in which wild-type F1 or F1 enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b ST34-156). With five different F1 enzymes, K d(bST34-156) ranged from 91 to 157 nM. Binding was strongly Mg2+-dependent; in EDTA buffer, Kd(b ST34-156) was increased to 1.25 μM. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of δ subunit to δ-depleted F1. The apparent K d(bST34-156) for this effect was increased from 150 nM in Mg2+ buffer to 1.36 μM in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F1 contributes to stator resistance and emphasizes the importance of Mg2+ in stator interactions.

Original languageEnglish
Pages (from-to)11253-11258
Number of pages6
JournalJournal of Biological Chemistry
Issue number12
StatePublished - Mar 19 2004


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