E. coli F₁F₀ ATP synthase, the central energy transduction enzyme, catalyzes the proton-driven ATP synthesis and ATP hydrolysis-driven proton translocation. The ϵ subunit, which is a part of F₁, plays a crucial role in binding F₁ to F₀, together with the γ subunit.

It is a two-domain protein in which the N-terminal domain is a 10-stranded β-sandwich and the C-terminal domain is a pair of α-helices in an anti-parallel coiled coil. ϵ is known to undergo conformational changes in response to the binding of nucleotides at the catalytic sites. These changes involve movement and unpacking of the C-terminal α-helices. In the "up"-state the helices are extended towards the α and β subunits of F ₁and in the "down"-state they are packed against the N-terminal domain of ϵ.

In this study, site-specific mutagenesis was performed to introduce unique cysteines at various positions of ϵ subunit. The accessibility of the cysteine residues was probed by labeling with N-maleimidylpropionyl biocytin (MPB). The results indicated that lot of small positional changes occur in the β-sheet region along the γ-ϵ-c interface. Labeling also showed that the cysteine residues in the C-terminal α-helices labeled well under all conditions, which means that the α-helices must open up, at least transiently. The accessibility pattern of the corresponding residues in the isolated ϵ and γ'-ϵ co-crystal structures does not match the labeling results obtained here. Effect of inhibition by ATP + MgSO ₄ and ADP-AlF₄ on the labeling were also studied. Most of the effect was found to be in the N-terminal region, especially near the γ-ϵ interface. This means that the conformational changes observed here are mostly at the γ-ϵ and γ-ϵ-c interfaces (regions interacting with other subunits), supporting the idea that the binding of nucleotides at the catalytic sites causes conformational changes in ϵ through the γ subunit. ATP did not have much effect on the residues in the α-helical region. This means that in the resting state the ϵ subunit spends some of its time in an intermediate conformation, in which most of the C-terminal residues are exposed.