Xiaolong Liu, Xiaoai Zhang, Yuanbo Cui, Jiachang Yue, Zhiyong Luo, Peidong Jiang
The National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Biochem Biophys Res Commun. 2006 Sep 1;347(3):752-7. doi: 10.1016/j.bbrc.2006.06.160. Epub 2006 Jul 7.
In order to observe mechanically driven proton flux in F(0)F(1)-ATPase coupled with artificial driven rotation on F(1) simultaneously, a double channel observation system was established. An artificial delta-free F(0)F(1)-ATPase was constructed with alpha(3), beta(3), epsilon, gamma, and c(n) subunits as rotator and a, b(2) as stator. The chromatophore was immobilized on the glass surface through biotin-streptavidin-biotin system, and the magnetic bead was attached to the beta subunit of delta-free F(0)F(1)-ATPase. The mechanically driven proton flux was indicated by the fluorescence intensity change of fluorescein reference standard (F1300) and recorded by a cooled digital CCD camera. The mechanochemical coupling stoichiometry between F(0) and F(1) is about 4.15 +/- 0.2H(+)/rev when the magnetic field rotated at 0.33 Hz (rps).
为了同时观察与F₁上人工驱动旋转相耦合的F₀F₁ - ATP酶中的机械驱动质子通量，建立了双通道观测系统。构建了一种无δ的人工F₀F₁ - ATP酶，以α₃、β₃、ε、γ和cₙ亚基作为转子，a、b₂作为定子。通过生物素 - 链霉亲和素 - 生物素系统将载色体固定在玻璃表面，并将磁珠连接到无δ的F₀F₁ - ATP酶的β亚基上。机械驱动的质子通量通过荧光素参考标准品（F1300）的荧光强度变化来指示，并由冷却的数字CCD相机记录。当磁场以0.33 Hz（转/秒）旋转时，F₀和F₁之间的机械化学耦合化学计量比约为4.15±0.2H⁺/转。
