F‐ATPase: specific observation of the rotating c subunit oligomer of EF_oEF₁

Abstract

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F o , of ATP synthase, F o F 1 , is still under contention. It was studied with EF o EF 1 ( 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. EF o EF 1 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 μm 2 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 F 1 and F o .