通过FRET监测活细菌中单个FoF1-ATP合酶的亚基旋转
Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET
摘要：
FoF 1-ATP合酶是线粒体、叶绿体和细菌中普遍存在的膜结合酶，其为细胞过程提供“化学能量货币”三磷酸腺苷（ATP）。在大肠杆菌中，ATP合成由质子动力（PMF）驱动，该质子动力包括质子浓度差ΔpH加上跨脂质膜的电势Δ pH。单分子体外实验已经证实，质子驱动的FoF 1-ATP合酶内的亚基旋转与ATP的合成。基于单分子荧光共振能量转移（FRET）的分子内距离测量的亚基旋转的动力学和不同的转子部分的步长已经解开。然而，这些实验是在由最大ΔpH ~ 4和未知Δ pH组成的PMF存在下完成的。相比之下，在活细菌中，跨质膜的最大ΔpH可能为0.75，并且Δ pH在-80 mV和-140 mV之间测量。因此，必须分别解决单个FoF 1-ATP酶的体内催化转换速率或体内旋转速度的问题。此外，必须确定维持高ATP水平所需的单个细菌中功能酶的绝对数量。我们报告了我们的进展，在体外和体内，这是一个新的标记方法，单分子FRET测量单FoF 1-ATP酶的亚基旋转测量。
英文摘要：
FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.
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