F-ATPase: specific observation of the rotating c subunit oligomer of EFoEF1

The rotary motion in response to ATP hydrolysis of the ring of c subunits o f the membrane portion, F-o, of ATP synthase, FoF1, is still under contenti on. It mas studied with EFoEF1 (Escherichia coli) using microvideograph wit h a fluorescent actin filament, To overcome the limited specificity of acti n attachment through a Cys-maleimide couple which might have hampered the i nterpretation of previous work, we engineered a 'strep-tag' sequence into t he 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. E FoEF1 was immobilized on a Ni-NTA-coated glass slide by the engineered His- tag at the N-terminus of subunit beta, In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 mu m(2) size, in same cases yielding a proportion of 5% rotating over total filaments. The rotation mas unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis f or ongoing studies on torque and angular elastic distortions between F-1 an d F-o. (C) 2000 Federation of European Biochemical Societies.