通过ABELtrap监测溶液中操纵的脂质体中的单膜蛋白动力学
Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap
摘要：
FoF 1-ATP合酶是维持细胞内三磷酸腺苷（ATP）水平的必需膜酶，由两个旋转马达组成。我们测量亚基旋转FoF 1-ATP合酶的分子内Foerster共振能量转移（FRET）在转子和定子的酶之间的两个荧光团。自由扩散的单酶在脂质囊泡的共焦FRET测量被限制到数百毫秒的通过激光焦点的渡越时间。我们评估了两种不同的方法来捕获共聚焦体积内的酶，以延长观察时间。Monte Carlo模拟结果表明，低激光功率的光镊不适用于直径为130 nm的脂质囊泡。A. E. Cohen（Harvard）和W. E. Moerner（Stanford）最近开发了一种反布朗电动势阱（ABELtrap），其能够明显地捕获溶液中的单个分子、蛋白质、病毒或囊泡。荧光颗粒的捕获是通过将实时、位置相关的反馈施加到微流体装置中的四个电极来实现的。在ABEL陷阱中，与给定目标位置的标准偏差小于200 nm。我们开发了一种ABELtrap与共聚焦FRET测量的组合，通过FRET在溶液中监测单膜酶动力学超过10秒。
英文摘要：
FoF1-ATP synthase is the essential membrane enzyme maintaining the cellular level of adenosine triphosphate (ATP) and comprises two rotary motors. We measure subunit rotation in FoF1-ATP synthase by intramolecular Foerster resonance energy transfer (FRET) between two fluorophores at the rotor and at the stator of the enzyme. Confocal FRET measurements of freely diffusing single enzymes in lipid vesicles are limited to hundreds of milliseconds by the transit times through the laser focus. We evaluate two different methods to trap the enzyme inside the confocal volume in order to extend the observation times. Monte Carlo simulations show that optical tweezers with low laser power are not suitable for lipid vesicles with a diameter of 130 nm. A. E. Cohen (Harvard) and W. E. Moerner (Stanford) have recently developed an Anti-Brownian electrokinetic trap (ABELtrap) which is capable to apparently immobilize single molecules, proteins, viruses or vesicles in solution. Trapping of fluorescent particles is achieved by applying a real time, position-dependent feedback to four electrodes in a microfluidic device. The standard deviation from a given target position in the ABELtrap is smaller than 200 nm. We develop a combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution.
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