Powering the cell: high resolution single-molecule investigation the mechanism of ATP synthesis

为细胞提供动力：高分辨率单分子研究 ATP 合成机制

• 批准号: BB/L01985X/1
• 负责人: Richard Berry
• 金额: $ 51.75万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL01985X%2F1
• 关键词: Powering; cell; resolution; single; molecule

All living cells are surrounded by a cell membrane, made of a lipid bilayer two molecules thick containing among other things protein molecules that span the membrane. Proteins are the molecules that make up most of the molecular machinery that performs the basic chemical and physical processes of life. Cells form and maintain gradients of charged ions (H+ or Na+) across the membrane by pumping them across, using energy derived from sunlight or food. These gradients are used to power the cells' metabolism. Some processes use the ion gradients directly, but in most cases the cell converts their energy into a chemical form by synthesizing the high-energy molecule adenosine triphosphate (ATP) from its breakdown products adenosine diphosphate (ADP) and inorganic phosphate (Pi). A transmembrane motor protein called F1FO ATP synthase performs this conversion for most of the ATP made by most organisms. F1FO is best understood as two rotary motors, F1 and FO, with linked rotors and stators. The motors generate torque in opposite directions so that the overall direction of rotation depends on which motor generates more torque. Usually FO, which is driven by the ion gradient, is stronger and it forces F1 backwards. F1 like many other molecular machines runs on ATP, and when driven backwards it works in reverse to make ATP. This is how most ATP is synthesized.F1 is one of the best understood of all molecular motors. Isolated F1 works via a mechanism that has been revealed by two decades of groundbreaking structural and single molecule studies. Its rotor rotates counterclockwise, taking 3 steps per rev, each step coupled to the use of on molecule of ATP fuel. Experiments tracking gold particles tens of nanometers across attached to the rotor, at several thousands of video frames per second, have shown the pattern of this rotation and how it depends on the nature and state of the fuel and the motor. But isolated F1 is not found in cells. FO is much more difficult to study than F1 because it is unstable without the membrane. A further limitation is that ATP synthesis by F1FO requires electrical separation between the opposite sides of an intact membrane and the means to produce and maintain an ion gradient. Traditional approaches to providing a membrane environment have had limited success in observing the rotation of F1FO.The aim of our project is to develop new approaches to providing a membrane environment, and to use them to understand mechanism of ATP synthesis by F1FO. We will develop two novel technologies for reconstitution of large membrane proteins into closed, well energized lipid bilayers amenable to high numerical resolution light microscopy, and explore their use in high-resolution single molecule measurements of the rotation of gold nanoparticle labels attached to fully functional F1FO and to isolated FO. We will use H+-coupled F1FO from Escherichia coli, which is a model organism, and if possible also H+- and Na+-coupled F1FO from Iliobacter tartaricus and various other bacterial species.

所有的活细胞都被细胞膜包围着，细胞膜由两分子厚的脂质双分子层组成，其中包括跨越细胞膜的蛋白质分子。蛋白质是构成大多数分子机制的分子，这些分子机制执行生命的基本化学和物理过程。细胞利用来自阳光或食物的能量，通过泵送带电离子（H+或Na+）穿过细胞膜，形成并维持带电离子（H+或Na+）的梯度。这些梯度被用来为细胞的新陈代谢提供能量。有些过程直接使用离子梯度，但在大多数情况下，细胞通过其分解产物二磷酸腺苷（ADP）和无机磷酸盐（Pi）合成高能分子三磷酸腺苷（ATP），将其能量转化为化学形式。一种叫做F1FO ATP合成酶的跨膜运动蛋白对大多数生物体产生的大部分ATP进行这种转化。F1FO最好理解为两个旋转电机，F1和FO，转子和定子相连。两个电机在相反的方向上产生转矩，因此整个旋转方向取决于哪个电机产生更大的转矩。通常由离子梯度驱动的FO更强，它迫使F1向后。F1像许多其他分子机器一样依靠ATP运转，当它被反向驱动时，它会反向工作以制造ATP。大多数ATP就是这样合成的。F1是所有分子马达中最容易理解的一种。经过20年开创性的结构和单分子研究，分离的F1通过一种机制发挥作用。它的转子逆时针旋转，每转3步，每步耦合到一分子ATP燃料的使用。实验以每秒数千个视频帧的速度跟踪附着在转子上的几十纳米的金颗粒，显示了这种旋转的模式，以及它是如何依赖于燃料和马达的性质和状态的。但是在细胞中没有发现孤立的F1。FO比F1更难研究，因为没有膜它是不稳定的。进一步的限制是，通过F1FO合成ATP需要在完整膜的两侧之间进行电分离，并需要产生和维持离子梯度的手段。提供膜环境的传统方法在观察F1FO的旋转方面取得了有限的成功。我们项目的目的是开发提供膜环境的新方法，并利用它们来了解F1FO合成ATP的机制。我们将开发两种新技术，用于将大膜蛋白重构成封闭的、能量充沛的脂质双分子层，适用于高数值分辨率光学显微镜，并探索它们在高分辨率单分子测量中应用，以测量附着在全功能F1FO和分离FO上的金纳米颗粒标签的旋转。我们将使用来自大肠杆菌的H+偶联F1FO，这是一种模式生物，如果可能的话，还将使用来自酒石酸杆菌和其他各种细菌的H+-和Na+偶联F1FO。

项目成果

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

• DOI: 10.3791/56909
• 发表时间: 2018-04-05
• 期刊: Journal of visualized experiments : JoVE
• 作者: Galkin MA;Russell AN;Vik SB;Berry RM;Ishmukhametov RR
• 通讯作者: Ishmukhametov RR

Comparison between single-molecule and X-ray crystallography data on yeast F1-ATPase.

• DOI: 10.1038/srep08773
• 发表时间: 2015-03-10
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Steel BC;Nord AL;Wang Y;Pagadala V;Mueller DM;Berry RM
• 通讯作者: Berry RM

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

一种简单的低成本设备可在标准光显微镜上采用四种表弹技术。

• DOI: 10.1038/srep20729
• 发表时间: 2016-02-08
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Ishmukhametov RR;Russell AN;Wheeler RJ;Nord AL;Berry RM
• 通讯作者: Berry RM

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes.

• DOI: 10.1038/ncomms13025
• 发表时间: 2016-10-06
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Ishmukhametov, Robert R.;Russell, Aidan N.;Berry, Richard M.
• 通讯作者: Berry, Richard M.