High resolution single-molecule observation of functional F1FO complex

细胞内许多重要的生物学过程都需要能量，而能量传递只是细胞膜上许多重要的生物学过程之一。生物能量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F1 FO由两个互补的旋转马达-跨膜FO和水溶性F1 -与连接的转子和连接的定子组成。虽然F1的旋转机制已经在单分子水平上表征[1]，FO需要在支持电化学梯度的密封脂质双层中重构，这意味着它还没有得到很好的理解。在受控的膜电压和相关分子浓度下对功能性F1 FO复合物进行分子观察。我们的方法囊泡融合[3]使用具有与靶膜相反电荷的脂质的蛋白脂质体实现膜蛋白到脂质双层中的递送。我们最近证明，这种快速的一步递送方法可用于整合大型脆弱的完整膜蛋白而不丧失功能，并将其与其他选定的膜蛋白混合[3]。具体地说，我们将F1 FO ATP合酶与代谢质子泵混合在几种不同的脂质双层结构中，所有脂质双层结构随后都合成ATP。水凝胶双层上的液滴（DHB）[4]在油包脂质溶液中仅使用纳升水滴，我们可以将联合收割机单分子观察和完整膜蛋白的跟踪与DHB的电压钳位和电流记录结合起来[4]囊泡融合第一次提供了将任何膜蛋白原则上递送到DHB中的能力，这与仅递送高度稳健的自掺入毒素α-溶血素的早期实验相反。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部液体环境的选项。Berry实验室最近建立了一种用于将F1 FO重组为蛋白脂质体[5]并随后将其递送到氢双层上的液滴中的超快速方案。F1 FO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，同时质子，钠离子，ATP，ADP和Pi分子的浓度完全受控。此外，该方法将允许我们第一次直接控制跨双分子层的电压并观察随之而来的旋转。该项目属于EPSRC生物物理学和软物质物理学研究领域的福尔斯。该项目由罗氏公司的Mathew Pletcher博士和Jeffrey爱马仕博士共同监督。参考文献[1]等人。Philos Trans R Soc Lond B Biol Sci，2013年。368（1611）：第20120023页。[2]Gao，Y.Q.等Cell，2005. 123（2）：第195-205页。[3]伊什穆哈梅托夫，R.，Nat Commun，2016. 7，13025 doi：10.1038/ncomms13025. [4]海伦，A. J.，等人J Am Chem Soc，2009. 131（5）：p. 1652-3. [5]伊什穆哈梅托夫，R.，等Biochim Biophys Acta，2005. 1706（1-2）：p. 110-6.

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.