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F‐ATPase: specific observation of the rotating c subunit oligomer of EF o EF 1
Author(s) -
Pänke Oliver,
Gumbiowski Karin,
Junge Wolfgang,
Engelbrecht Siegfried
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01436-8
Subject(s) - oligomer , protein subunit , atpase , chemistry , biophysics , crystallography , biochemistry , enzyme , biology , polymer chemistry , gene
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 β. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 μm 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 .