重组FoF1-ATP合酶在溶液中的快速ATP依赖性亚基旋转
Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution
摘要：
FoF 1-ATP酶是一种广泛存在的膜结合的旋转马达酶，可以催化ATP的合成和水解。它们的酶动力学受内部亚基旋转、底物和产物浓度、机械抑制机制以及质子跨膜的电化学电位控制。单分子Förster共振能量转移（smFRET）已被用于检测包埋在自由扩散脂质体中的FoF 1-ATP酶内的亚基旋转。我们现在报告，动力学监测功能旋转可以延长从毫秒到秒，利用反布朗电动势阱（ABEL陷阱）。这些延长的观察时间使我们能够观察溶液中单个FoF 1-脂质体的功能旋转的波动速率。ATP依赖的催化速率的广泛分布被揭示。质子的电化学势的积累被证实限制ATP水解的最大速率。在存在离子载体或解偶联剂的情况下，在单个重构FoF 1-ATP酶中测得的最快亚基旋转速度为每秒180个完整轮。这是更快地比生化系综平均测量，但没有那么快的最大转速之前报道的分离的单个F1复合物从膜包埋的Fo复合物解偶联。ABEL陷阱测量的进一步应用应有助于解决这种波动率的亚基旋转的机械原因。
英文摘要：
FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. Single-molecule Förster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.
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