使用快速抗布朗电动陷阱与用于单分子FRET的FoF1-ATP合酶融合的优化的绿色荧光蛋白
Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap
摘要：
自由扩散的单分子在溶液中的观察时间受到所附荧光标记物的光物理学和足够的信号背景比所需的飞升范围内的小观察体积的限制。为了通过共焦检测体积延长扩散限制的观察时间，A。E. Cohen和W. E. Moerner发明并建造了ABELtrap -一种微流体装置，可以用电动陷阱主动抵消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芯片控制。这种ABELtrap可将单个荧光纳米颗粒保持100秒以上，与自由扩散相比，荧光纳米颗粒的观察时间增加了10000倍。为了实时监测单个膜蛋白的构象变化，我们使用Förster共振能量转移（smFRET）记录了两种特异性连接的染料之间的连续距离变化。FoF 1-ATP合酶的α-亚基与mNeonGreen融合导致在较低激光激发功率下改善的信号背景比。这增加了我们测量的蛋白脂质体的陷阱持续时间超过2秒。此外，我们观察到不同的smFRET水平归因于FRET供体（mNeonGreen）和受体（Alexa 568）荧光团之间的不同距离分别连接在FoF 1-ATP合酶的a-和c-亚基。
英文摘要：
Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap - a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. 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 chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.
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。
