The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, Fo, of ATP synthase, FoF1, is still under contention. It was studied with EFoEF1 (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys-maleimide couple which might have hampered the interpretation of previous work, we engineered a ‘strep-tag’ sequence into the C-terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EFoEF1 was immobilized on a Ni-NTA-coated glass slide by the engineered His-tag at the N-terminus of subunit β. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 μm2 size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F1 and Fo.
