Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.
Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.
复制标题
单酶的时间依赖性 FRET：H( )-ATP 合酶中的结构域运动和催化作用。
作者：
R. Bienert;B. Zimmermann;Verena Rombach;P. Gräber
H(+)-ATP 合酶是通过旋转机制将跨膜质子运输与 ADP 和无机磷酸盐合成 ATP 耦合的分子机器。单分子中的单对荧光共振能量转移（spFRET）是分析构象变化的有力工具。它用于研究大肠杆菌 (EF(0)F(1)) 和菠菜叶绿体 (CF(0)F(1)) 的 H(+)-ATP 合酶在催化过程中的亚基运动。这些酶被整合到脂质体膜中，从而产生跨膜 pH 值差异，这是 ATP 合成所必需的。用荧光供体和受体标记不同亚基上的适当位点后，测量 spFRET 的动力学。对 E(FRET) 迹线的分析揭示了 ATP 合成和 ATP 水解过程中 ε 和 γ 亚基以 120° 步长、方向相反的旋转运动。步进运动的特点是 120° 步进速度快于 1 ms，随后是平均停留时间为 15 ms 的休息期，这与酶的周转时间一致。除了催化过程中的三种构象外，还发现了催化后观察到的非活性构象。
H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.
DOI：
10.1073/pnas.96.7.3670
发表时间：
1999-03-30
影响因子：
11.1
作者：
Deniz, AA;Dahan, M;Schultz, PG
通讯作者：
Schultz, PG
DOI：
10.1016/s0005-2728(98)00048-6
发表时间：
1998-06
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
S. Wilkens;R. Capaldi
通讯作者：
S. Wilkens;R. Capaldi
DOI：
10.1016/s0021-9258(18)43822-7
发表时间：
1994-12
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Steven B. VikS;B. Antonio
通讯作者：
Steven B. VikS;B. Antonio