F‐ATPase: specific observation of the rotating c subunit oligomer of EFoEF1
F‐ATPase: specific observation of the rotating c subunit oligomer of EFoEF1
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DOI：
10.1016/s0014-5793(00)01436-8
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发表时间：
2000-04
期刊：
影响因子：
3.5
通讯作者：
O. Pänke;Karin Gumbiowski;W. Junge;S. Engelbrecht
中科院分区：
文献类型：
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作者：
O. Pänke;Karin Gumbiowski;W. Junge;S. Engelbrecht
ATP合酶FoF 1的膜部分Fo的c亚基环响应ATP水解的旋转运动仍有争议。用荧光肌动蛋白丝的显微摄像研究了EFoEF 1（大肠杆菌）。为了克服通过Cys-马来酰亚胺偶联的肌动蛋白附着的有限特异性，这可能妨碍了对先前工作的解释，我们将“strep-标签”序列工程化到亚基c的C末端。它用于（a）纯化全酶和（B）单特异性地将荧光肌动蛋白丝连接到亚基c。将EFoEF 1固定在Ni-NTA包被的载玻片上，并在β亚基N端引入His标签。在MgATP存在的情况下，我们观察到每幅2000 μ m2大小的图片中有多达5条逆时针旋转的肌动蛋白丝，在某些情况下，旋转的比例占总丝的5%。该旋转明确归因于亚基c的环。新的，双重工程结构作为一个更坚实的基础，正在进行的研究扭矩和角弹性变形之间的F1和Fo。
The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, Fo, of ATP synthase, FoF1, is still under contention. It was studied with EFoEF1(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. EFoEF1was 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 μm2size, 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 F1and Fo.