ABEL Trap中单分子FRET对F1-ATPase调控开关的研究
The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap
摘要：
F1-ATP酶是膜包埋酶FoF 1-ATP合酶的可溶性部分，其催化真核细胞和真细菌细胞中三磷酸腺苷的产生。相反，F1部分也可以在三个催化结合位点快速水解ATP。因此，必须在细胞中最小化F1（或FoF 1）对“非生产性”ATP水解的催化作用。在细菌中，ATP亚基被认为通过机械地将其C末端插入F1的旋转马达区域来控制和阻断ATP水解。我们研究了这一拟议的机制，通过标记F1特异性与两个荧光团监测的C-末端的Förster共振能量转移的α亚基。单个F1分子被反布朗电动势阱捕获在溶液中，该反布朗电动势阱将FRET标记的F1保持在原位数百毫秒的延长观察时间，受光漂白的限制。比较不同生化条件下单个F1和FRET直方图的FRET变化，以评估拟议的调控机制。
英文摘要：
F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the epsilon subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the epsilon subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.
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