High resolution single-molecule observation of functional F1FO complex

功能性 F1FO 复合物的高分辨率单分子观察

• 批准号: 1939972
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1939972
• 关键词: resolution; single; molecule; observation; functional

BackgroundMany crucial biological processes in cells require energy, and energy transduction is only one of the many important biological processes that are located at cell membranes. The central process in bioenergetics is the synthesis of high-energy ATP molecules using a transmembrane electrochemical gradient called the Protonmotive Force. ATP-synthase F1FO consists of two complementary rotary motors - transmembrane FO and water-soluble F1 - with linked rotors and linked stators. While the rotational mechanism of F1 has been characterized on the single molecule level [1], the requirement for FO to be reconstituted in a sealed lipid bilayer supporting an electrochemical gradient means that it is much less well understood.The aim of this project will be to achieve high resolution single-molecule observation of functional F1FO complex under controlled membrane voltage and concentration of relevant molecules.Our MethodsVesicle fusion [3]Delivery of membrane proteins into a lipid bilayer is achieved using proteoliposomes with lipids of opposite charge to the target membrane. We recently demonstrated that this fast, one-step delivery method can be used for incorporating large, fragile integral membrane proteins without loss of function, and mixing them with other chosen membrane proteins [3]. Specifically, we mixed F1FO ATP-synthase with a metabolic proton pump in several different lipid bilayer configurations all of which subsequently synthesized ATP.Droplet on Hydrogel Bilayer (DHB) [4]Using only a nanoliter water droplet in a lipid-in-oil solution, we can combine single-molecule observation and tracking of integral membrane proteins with voltage clamping of the DHB and current recordings [4].Vesicle fusion offers for the first time the ability to deliver in principle any membrane protein into a DHB, in contrast to earlier experiments where only the highly robust self-incorporating toxin a-hemolysin was delivered. Unpublished developments in the Berry lab have added the option of perfusion of the liquid environment inside the droplet using a microfluidic device.An ultrafast protocol for reconstituting F1FO into proteoliposomes [5] and their consequent delivery into Droplet on Hydrogen Bilayers was recently established in the Berry lab. F1FO can be labeled with a gold nanoparticle and its rotation tracked with ultra-high angular and temporal resolution, while concentrations of protons, sodium ions, ATP, ADP and Pi molecules are fully controlled. Moreover, the method will allow us for the first time to directly control the voltage across the bilayer and observe consequent rotation.This project falls within the EPSRC Biophysics and Soft Matter Physics research area. The project is co-supervised by Dr Mathew Pletcher and Dr Jeffrey Hermes from Roche.References[1] Bilyard, T., et al. Philos Trans R Soc Lond B Biol Sci, 2013. 368(1611): p. 20120023.[2] Gao, Y.Q. et al. Cell, 2005. 123(2): p. 195-205.[3] Ishmukhametov, R., Nat Commun, 2016. 7, 13025 doi: 10.1038/ncomms13025.[4] Heron, A.J., et al. J Am Chem Soc, 2009. 131(5): p. 1652-3.[5] Ishmukhametov, R., et al. Biochim Biophys Acta, 2005. 1706(1-2): p. 110-6.

背景细胞中许多重要的生物过程都需要能量，而能量转导只是位于细胞膜的许多重要生物过程之一。生物能学的核心过程是利用称为质子动力的跨膜电化学梯度合成高能 ATP 分子。 ATP 合酶 F1FO 由两个互补的旋转马达（跨膜 FO 和水溶性 F1）组成，并具有相连的转子和相连的定子。虽然 F1 的旋转机制已在单分子水平上进行了表征 [1]，但需要在支持电化学梯度的密封脂质双层中重构 FO，这意味着人们对它的了解还少得多。该项目的目的是在受控膜电压和相关分子浓度下实现功能性 F1FO 复合物的高分辨率单分子观察。 方法囊泡融合[3]使用具有与目标膜电荷相反的脂质的蛋白脂质体来将膜蛋白递送到脂质双层中。我们最近证明，这种快速、一步的递送方法可用于整合大的、脆弱的整合膜蛋白而不丧失功能，并将它们与其他选定的膜蛋白混合[3]。具体来说，我们将 F1FO ATP 合酶与代谢质子泵在几种不同的脂质双层配置中混合，所有这些随后都合成了 ATP。水凝胶双层 (DHB) 上的液滴 [4]仅在油包脂溶液中使用纳升水滴，我们可以将单分子观察和完整膜蛋白的跟踪与电压结合起来 DHB 和当前记录的钳位 [4]。囊泡融合原则上首次提供了将任何膜蛋白递送到 DHB 中的能力，这与早期的实验相反，早期的实验仅递送高度稳健的自掺入毒素 a-溶血素。 Berry 实验室未发表的进展增加了使用微流体装置灌注液滴内液体环境的选项。Berry 实验室最近建立了一种超快方案，用于将 F1FO 重组为蛋白脂质体 [5]，然后将其输送到氢双层上的 Droplet。 F1FO 可以用金纳米颗粒标记，并以超高的角度和时间分辨率跟踪其旋转，同时完全控制质子、钠离子、ATP、ADP 和 Pi 分子的浓度。此外，该方法将使我们首次能够直接控制双层的电压并观察随后的旋转。该项目属于 EPSRC 生物物理学和软物质物理学研究领域。该项目由罗氏公司的 Mathew Pletcher 博士和 Jeffrey Hermes 博士共同监督。参考文献[1] Bilyard, T. 等人。 Philos Trans R Soc Lond B Biol Sci，2013。368（1611）：第。 20120023.[2]高YQ.等人。细胞，2005。123(2)：p。 195-205。[3] Ishmukhametov, R., Nat Commun, 2016. 7, 13025 doi: 10.1038/ncomms13025.[4]海伦，A.J.，等人。美国化学学会杂志，2009 年。131(5)：第 14 页。 1652-3.[5]伊什穆哈梅托夫，R.，等人。 Biochim 生物物理学学报，2005 年。1706(1-2)：第 19 页。 110-6。