Monitoring the rotary mechanism of a single FoF1-ATP synthase in the ABELtrap

FoF1-ATP合成酶通过亚基内部旋转的机械化学机制产生并维持细胞内的[ATP]水平。我们研究了单个酶的F1部分的γ和ε亚基和Fo部分的c环的逐步旋转。用不同的荧光团特异性标记两个亚基，利用Förster共振能量转移（FRET）监测旋转过程中的内部距离变化。我们的共聚焦单分子FRET方法使用了在直径约120nm的脂质体中重组的自由扩散酶。由于检测体积的限制，蛋白脂质体的平均传递时间为30 ms，我们可以研究在1 mM ATP下ATP水解和高Delta_ph和Delta_psi下ATP合成时的亚基旋转。斯坦福大学的w·e·莫尔纳（W. E. Moerner）小组开发了一种反布朗电动陷阱（ABELtrap），它可以在微流体装置中保持小荧光颗粒（即蛋白质）长达10秒。ABELtrap中的单分子FRET测量允许回答在较低[ATP] ATP水解过程中epsilon旋转120°的可能子步骤的开放机制问题，以澄清连续36°c步骤的相同或不同停留时间，并使用适当的单分子统计来测量两个耦合转子亚基的内部弹性变形。申请人作为斯坦福大学访问学者参加这些ABELtrap实验。

FoF1-ATP synthases produce and maintain the [ATP] level in cells by a mechanochemical mechanism using internal rotation of subunits. We have investigated the stepwise rotation of the gamma and epsilon subunits in the F1 part and of the c-ring in the Fo part of single enzymes. Two subunits were labeled specifically with different fluorophores to monitor the internal distance changes during rotation using Förster resonance energy transfer (FRET). Our confocal single-molecule FRET approach used freely diffusing enzymes reconstituted in liposomes with a diameter of about 120 nm. Limited by a mean transit time of 30 ms for the proteoliposomes through the detection volume, we could study subunit rotation during ATP hydrolysis at 1 mM ATP and ATP synthesis at high Delta_ph and Delta_psi only.The group of W. E. Moerner in Stanford has developed an Anti-Brownian electrokinetic trap (ABELtrap) which holds a small fluorescent particle (i.e. a protein) in place for up to 10 seconds in a microfluidic device. Single-molecule FRET measurements in the ABELtrap allow to answer the open mechanistic questions of possible substeps of 120° rotation of epsilon during ATP hydrolysis at lower [ATP], to clarify the identical or different dwell times of the sequential 36° steps of c and to measure the internal elastic deformation of the two coupled rotor subunits with appropriate single-molecule statistics. The applicant participates in these ABELtrap experiments as a Stanford Visting Scholar.