PMID:10781800
| Citation |
Pänke, O, Gumbiowski, K, Junge, W and Engelbrecht, S (2000) F-ATPase: specific observation of the rotating c subunit oligomer of EF(o)EF(1). FEBS Lett. 472:34-8 |
|---|---|
| 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 beta. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 microm(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). |
| Links | |
| Keywords |
Actins; Biotin; Catalytic Domain; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Fluorescent Dyes; Microscopy, Video; Oligopeptides/chemistry; Proton-Translocating ATPases/chemistry |
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