Folding and stability of the b subunit of the F1F0 ATP synthase

Abstract

The F₁F₀ ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F₀) and soluble (F₁) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F₁F₀ complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T_m values between 30 and 40°C. CD spectra for the region comprising residues 53–122 (b_53–122) showed θ₂₂₂/θ₂₀₈ = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b_53–122 (ΔG, ΔH and ΔC_p) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.