F-ATPase: specific observation of the rotating c subunit oligomer of EFoEF1
Autor(en): | Panke, O Gumbiowski, K Junge, W Engelbrecht, S |
Stichwörter: | ATP synthase; BETA-SUBUNIT; Biochemistry & Molecular Biology; Biophysics; Cell Biology; CROSS-LINKING; DIRECTED MUTAGENESIS; EFoEF1; ELASTIC ENERGY; EPSILON-SUBUNIT; ESCHERICHIA-COLI F1-ATPASE; F-1-ATPASE; GAMMA-SUBUNIT; PURIFICATION; rotation; single molecule; subunit c; SYNTHASE | Erscheinungsdatum: | 2000 | Herausgeber: | ELSEVIER SCIENCE BV | Journal: | FEBS LETTERS | Volumen: | 472 | Ausgabe: | 1 | Startseite: | 34 | Seitenende: | 38 | Zusammenfassung: | The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F-o, of ATP synthase, FoF1, is still under contention. It mas studied with EFoEF1 (Escherichia coli) using microvideograph 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 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 for ongoing studies on torque and angular elastic distortions between F-1 and F-o. (C) 2000 Federation of European Biochemical Societies. |
ISSN: | 00145793 | DOI: | 10.1016/S0014-5793(00)01436-8 |
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geprüft am 06.05.2024