High resolution single-molecule observation of functional F1FO complex

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上的许多重要生物过程之一。生物能量学的核心过程是利用称为质子动势的跨膜电化学梯度合成高能ATP分子。atp合成酶F1FO由两个互补的旋转马达组成-跨膜FO和水溶性F1 -具有连接的转子和连接的定子。虽然F1的旋转机制已经在单分子水平上进行了表征，但FO需要在支持电化学梯度的密封脂质双分子层中重构，这意味着人们对其知之甚少。本项目的目标是在可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。我们的方法：囊泡融合[3]利用与靶膜带相反电荷的脂质蛋白脂质体将膜蛋白递送到脂质双分子层。我们最近证明，这种快速的一步递送方法可用于在不丧失功能的情况下整合大型脆弱的整体膜蛋白，并将其与其他选定的膜蛋白[3]混合。具体来说，我们将F1FO ATP合成酶与代谢质子泵混合在几种不同的脂质双分子层构型中，所有这些构型随后都合成了ATP。只需在油中脂溶液中使用一纳升的水滴，我们就可以将单分子观察和整体膜蛋白的跟踪与DHB的电压夹紧和电流记录结合起来。囊泡融合首次提供了原则上将任何膜蛋白传递到DHB的能力，而不是像早期的实验那样只传递高度强大的自结合毒素a-溶血素。Berry实验室未发表的研究成果增加了使用微流体装置在液滴内灌注液体环境的选项。最近在Berry实验室建立了一种将F1FO重组为蛋白质脂质体[5]并随后将其递送到氢双分子层上的液滴的超快速方案。F1FO可以用金纳米粒子标记，其旋转可以用超高的角度和时间分辨率进行跟踪，同时质子、钠离子、ATP、ADP和Pi分子的浓度可以完全控制。此外，该方法将允许我们第一次直接控制双分子层上的电压并观察随之而来的旋转。该项目属于EPSRC生物物理和软物质物理研究领域。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bbb10 Bilyard， T.，等。中国生物医学工程学报，2013。368(11): p. 20120023.[2]高永强等。细胞,2005。21 (2): p. 195-205Ishmukhametov， R., Nat Commun, 2016。7, 13025 doi: 10.1038/ncomms13025.[4]赫伦，a.j.等人。化学学报，2009。131(5): p. 1652- 53伊什穆哈梅托夫，R.等。生物化学学报，2005。1706(1-2): 110-6页。

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.