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。我们研究了 F1 部分中的 γ 和 epsilon 亚基以及 Fo 部分中的 C 环的逐步旋转。两个亚基用不同的荧光团专门标记，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。 FRET 方法使用在直径约 120 nm 的脂质体中自由扩散的酶，由于蛋白脂质体通过检测体积的平均传输时间为 30 ms，我们可以研究 1 mM ATP 下的 ATP 水解和 1 mM ATP 下的 ATP 合成过程中的亚基旋转。仅高 Delta_ph 和 Delta_psi。斯坦福大学的 W. E. Moerner 小组开发了一种反布朗动电陷阱 (ABELtrap)，该陷阱具有小荧光颗粒（即蛋白质）在微流体装置中停留长达 10 秒 ABELtrap 中的单分子 FRET 测量可以回答 ATP 水解过程中 epsilon 120° 旋转可能的子步骤的开放机制问题。 ATP]，阐明 c 的连续 36° 步长的相同或不同停留时间，并用适当的单分子测量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了这些 ABELtrap 实验。