title: F-ATPase: specific observation of the rotating c subunit oligomer of EF(o)EF(1).
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).
authors.affiliation: Universität Osnabrück, Fachbereich Biologie, Abteilung Biophysik, Barbarastr. 11, 49069, Osnabrück, Germany.
authors.forename: O
authors.initials: O
authors.lastname: Pänke
authors.forename: K
authors.initials: K
authors.lastname: Gumbiowski
authors.forename: W
authors.initials: W
authors.lastname: Junge
authors.forename: S
authors.initials: S
authors.lastname: Engelbrecht
mesh_terms: D000199:Actins; D001710:Biotin; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D005456:Fluorescent Dyes; D018715:Microscopy, Video; D009842:Oligopeptides / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D001710:Biotin; D006180:Proton-Translocating ATPases