在反布朗电动势阱中监测单个FRET标记的FoF1-ATP合酶中的亚基旋转
Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap
摘要：
FoF 1-ATP合成酶是催化“生物能量货币”三磷酸腺苷（ATP）合成的膜蛋白。该酶利用内部亚基旋转将质子动力转化为化学键。我们应用单分子Förster共振能量转移（FRET）来监测两个耦合马达F1和Fo中的亚基旋转。因此，必须从大肠杆菌的质膜中分离酶，荧光标记并重组成120 nm大小的脂质囊泡以产生脂蛋白体。这些自由扩散的脂蛋白体偶尔穿过共聚焦检测体积，导致光子爆发。构象动力学的酶的FRET效率在一个单一的光子爆发的顺序变化进行了鉴定。通过在反布朗电动势阱（ABELtrap，由A. E. Cohen和W. E. Moerner）。在这里，我们描述了FoF 1-ATP合酶的制备程序和模拟FRET效率轨迹为“困”的蛋白脂质体。隐马尔可夫模型应用于信号与背景比的限制，用于识别旋转酶在低ATP浓度下运行时的停留和子步骤，由低激光功率激发，并由ABELtrap限制。
英文摘要：
FoF1-ATP synthase is the membrane protein catalyzing the synthesis of the 'biological energy currency' adenosine triphosphate (ATP). The enzyme uses internal subunit rotation for the mechanochemical conversion of a proton motive force to the chemical bond. We apply single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the two coupled motors F1 and Fo. Therefore, enzymes have to be isolated from the plasma membranes of Escherichia coli, fluorescently labeled and reconstituted into 120-nm sized lipid vesicles to yield proteoliposomes. These freely diffusing proteoliposomes occasionally traverse the confocal detection volume resulting in a burst of photons. Conformational dynamics of the enzyme are identified by sequential changes of FRET efficiencies within a single photon burst. The observation times can be extended by capturing single proteoliposomes in an anti-Brownian electrokinetic trap (ABELtrap, invented by A. E. Cohen and W. E. Moerner). Here we describe the preparation procedures of FoF1-ATP synthase and simulate FRET efficiency trajectories for 'trapped' proteoliposomes. Hidden Markov Models are applied at signal-to-background ratio limits for identifying the dwells and substeps of the rotary enzyme when running at low ATP concentrations, excited by low laser power, and confined by the ABELtrap.
机构、总结、中文摘要均由AI分析而来，可能存在错误，请以论文正文为准。
