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

监测 ABELtrap 中单个 FoF1-ATP 合酶的旋转机制

• 批准号: 230720104
• 负责人: Professor Dr. Michael Börsch
• 金额: --
• 依托单位: Department of Chemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/230720104?language=en
• 关键词: Monitoring; rotary; mechanism; single; FoF1

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.

FoF 1-ATP脱氢酶通过使用亚基内部旋转的机械化学机制产生并维持细胞中的[ATP]水平。我们已经研究了逐步旋转的γ和γ亚基的F1部分和C-环的Fo部分的单酶。用不同的荧光团特异性标记两个亚基，以使用福斯特共振能量转移（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方法使用在直径约120 nm的脂质体中重构的自由扩散酶。由于蛋白脂质体通过检测体积的平均通过时间为30 ms，我们只能研究在1 mM ATP下ATP水解和高Delta_ph和Delta_psi下ATP合成过程中的亚基旋转。E.斯坦福大学的Moerner开发了一种反布朗电动陷阱（ABELtrap），它可以在微流体装置中将一个小的荧光颗粒（即蛋白质）固定在适当的位置长达10秒。在ABELtrap中的单分子FRET测量允许回答在较低[ATP]下ATP水解过程中120°旋转的可能子步骤的开放机制问题，以澄清连续36°步骤c的相同或不同的停留时间，并测量具有适当单分子统计的两个耦合转子子单元的内部弹性变形。申请人作为斯坦福大学访问学者参与这些ABELtrap实验。