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

FEBS Letters

Junge, Wolfgang¹  Pänke, Oliver¹  Engelbrecht, Siegfried¹  Gumbiowski, Karin¹ 

[1] Universität Osnabrück, Fachbereich Biologie, Abteilung Biophysik, Barbarastr. 11, 49069 Osnabrück, Germany

关键词: ATP synthase;    Subunit c;    EFoEF1;    Single molecule;    Rotation;   

DOI  :  10.1016/S0014-5793(00)01436-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F_o, of ATP synthase, F_oF₁, is still under contention. It was studied with EF_oEF₁ (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_oEF₁ 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 F_o.

【 授权许可】

Unknown