High resolution single-molecule observation of functional F1FO complex

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上的许多重要生物过程之一。生物能量学的中心过程是利用称为质子力的跨膜电化学梯度合成高能的ATP分子。三磷酸腺苷合成酶F1FO由两个互补的旋转电机组成--跨膜FO和水溶性F1--它们的转子和定子相连。虽然F1的旋转机制已经在单分子水平上得到了表征[1]，但对于FO必须在支持电化学梯度的密封脂质双层中重组的要求意味着人们对它的了解要少得多。本项目的目标是在控制膜电压和相关分子浓度的情况下实现对功能F1FO复合体的高分辨率单分子观察。我们的方法囊泡融合[3]利用脂类与靶膜电荷相反的蛋白脂体将膜蛋白输送到脂双层中。我们最近证明，这种快速的一步递送方法可以用于整合大的、脆弱的完整膜蛋白而不会失去功能，并将它们与其他选定的膜蛋白混合[3]。具体地说，我们将F1FO ATP合成酶与几种不同脂质双层配置的代谢质子泵混合在一起，所有这些都随后合成了ATP。在水凝胶双层(DHB)[4]上，我们只使用油包脂溶液中的一个纳升水滴，就可以结合对完整膜蛋白的单分子观察和跟踪与DHB的电压钳制和电流记录[4]。囊泡融合首次提供了原则上将任何膜蛋白输送到DHB的能力，而不像以前的实验那样，只有高度强健的自结合毒素a-溶血素被输送。Berry实验室未发表的进展增加了使用微流控设备在液滴内部灌流液体环境的选项。Berry实验室最近建立了一种超快方案，用于将F1FO重组为蛋白质脂质体[5]，并随后将其输送到氢双层上的液滴中。可以用金纳米颗粒标记F1FO，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，该方法将首次使我们能够直接控制双分子层上的电压并观察随后的旋转。该项目属于EPSRC生物物理和软物质物理研究领域。该项目由来自罗奇的马修·普莱彻博士和杰弗里·赫尔墨斯博士共同监督。Philos Trans R Soc Lond B生物科学，2013。368(1611年)：20120023页。[2]高永全等人。细胞，2005年。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]Heron，A.J.等人。J am Chem Soc，2009。131(5)：1652-3页[5]Ishmukhametov，R.，et al.Bichim生物物理学报，2005。1706(1-2)：第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.