在快速反布朗电动势阱中观察单个FoF1-ATP酶的构象
Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap
摘要：
为了实时监测脂质体中单个膜转运蛋白的构象变化，我们将两个荧光团连接到蛋白质的选定结构域。染料之间的顺序距离变化记录和分析福斯特共振能量转移（FRET）。使用在脂质体中重构的自由扩散膜蛋白，观察时间受到通过共聚焦检测体积的布朗运动的限制。A. E. Cohen和W. E. Moerner发明并构建了微流体装置，以主动抵消电动陷阱（ABELtrap）中单个纳米颗粒的布朗运动。在这里，我们提出了一个版本的ABELtrap的激光聚焦模式产生的电光光束偏转器和可编程FPGA控制。这种ABEL陷阱可以容纳单个荧光纳米珠超过100秒，将单个颗粒的观察时间增加了1000倍以上。在ABELtrap中可以检测到单个FRET标记的膜酶FoF 1-ATP合酶的构象变化。
英文摘要：
To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Forster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices to actively counteract Brownian motion of single nanoparticles in electrokinetic traps (ABELtrap). Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA. This ABELtrap could hold single fluorescent nanobeads for more than 100 seconds, increasing the observation times of a single particle by more than a factor of 1000. Conformational changes of single FRET-labeled membrane enzymes FoF1-ATP synthase can be detected in the ABELtrap.
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