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.

FOF1-ATP合酶通过使用亚基的内部旋转的机械化学机制产生并维持细胞中的[ATP]水平。我们已经研究了单个酶的FO部分中F1部分和C形环中γ和Epsilon亚基的逐步旋转。使用Förster共振能量转移（FRET），用不同的荧光团特异性标记了两个亚基，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为120 nm的脂质体中自由扩散的酶。通过检测量，蛋白脂质体的平均运输时间限制为30 ms，我们可以研究ATP水解期间的亚基在1 mm ATP下的水解过程中的旋转，并且仅在High delta_ph和Delta_psi时进行ATP合成。在微流体设备中最多可放置10秒。 ABELTRAP中的单分子特品格测量值可以回答下[ATP]在ATP水解过程中epsilon可能取代的可能取代的开放机理问题，以阐明C的顺序36°C的相同或不同的停留时间，以测量两个耦合的旋转单个统计量的内部弹性变形。这些Abeltrap实验的申请人作为斯坦福大学徒步旅行学者。