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

FOF1-ATP合酶通过亚基内部旋转的机械力化学机制产生和维持细胞内的[ATP]水平。我们研究了单酶F1部分的γ和epsilon亚基和Fo部分的c-环的逐级旋转。使用Förster共振能量转移(FRET)技术，用不同的荧光团标记两个亚基，以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120 nm的脂质体中重组的自由扩散酶。受蛋白质脂质体通过检测体积的平均传输时间30ms的限制，我们只能研究1 mM ATP在ATP水解过程中的亚基旋转和在高Delta_ph和Delta_psi下的ATP合成。斯坦福大学的W.E.Moerner团队开发了一种反布朗电动陷阱(ABELTrap)，它在微流控设备中将一个小的荧光颗粒(即蛋白质)保持在适当的位置长达10秒。ABELTrap中的单分子FRET测量允许回答在较低[ATP]的ATP水解过程中epsilon可能的120°旋转的子步骤的开放的机制问题，澄清c的连续36°步骤的相同或不同的停留时间，并用适当的单分子统计来测量两个耦合的转子亚单位的内部弹性变形。申请者以斯坦福访问学者的身份参加了这些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.