Folding and stability of the b subunit of the F 1 F 0 ATP synthase

Abstract

The F 1 F 0 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 (F 0 ) and soluble (F 1 ) sectors. The three‐dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F 1 F 0 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 θ 222 /θ 208 = 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.