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.
Highlights
To quantitatively characterize binding of b subunit to the F1 ␣33 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
Until 1994, the consensus based on electron microscopy and biochemical studies was that F1 was bound to F0 by a single “central stalk” presumed to contain ␥, ⑀, ␦, and b subunits
We studied the effects of Mg2ϩ cation on the ability of the cytoplasmic portion of b subunit to promote binding of ␦ subunit to F1, and we introduced Trp residues into the b subunit at residues 92 and 109 to test the effects on interaction with F1
Summary
Until 1994, the consensus based on electron microscopy (for one example, see Ref. 6) and biochemical studies (for one example, see Ref. 7) was that F1 was bound to F0 by a single “central stalk” presumed to contain ␥, ⑀, ␦, and b subunits. The b subunit interacts with the C-terminal domain of ␦ (24 –26), with the C-terminal residues of the b subunit playing an important role. The addition of the cytoplasmic portion of the b subunit altered the conformation of the F1 catalytic sites as shown by spin-labeling studies [29]. These approaches could not quantitatively determine the strength of interaction between b and F1. We studied the effects of Mg2ϩ cation on the ability of the cytoplasmic portion of b subunit to promote binding of ␦ subunit to F1, and we introduced Trp residues into the b subunit at residues 92 and 109 to test the effects on interaction with F1
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