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

所有活细胞都被细胞膜包围，细胞膜由两个分子厚的脂质双层组成，其中包含跨越膜的蛋白质分子。蛋白质是组成大部分分子机器的分子，这些分子机器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，形成并保持带电离子（H+或Na+）的梯度。这些梯度用于为细胞的新陈代谢提供动力。一些过程直接使用离子梯度，但在大多数情况下，细胞通过从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。一种称为F1 FO ATP合成酶的跨膜马达蛋白完成了大多数生物体产生的大部分ATP的转换。F1 FO最好理解为两个旋转电机，F1和FO，转子和定子相连。电机在相反方向上产生扭矩，使得旋转的总体方向取决于哪个电机产生更多扭矩。通常，由离子梯度驱动的FO更强，并且它迫使F1向后。像许多其他分子机器一样，F1在ATP上运行，当向后驱动时，它反向工作以产生ATP。这就是大多数ATP合成的方式。F1是所有分子马达中最好理解的一个。分离的F1通过一种机制发挥作用，这种机制已经被二十年的突破性结构和单分子研究所揭示。它的转子逆时针旋转，每转3步，每一步都与ATP燃料分子的使用相耦合。以每秒数千帧的视频跟踪连接在转子上的数十纳米大小的金颗粒的实验显示了这种旋转的模式以及它如何取决于燃料和电机的性质和状态。但在细胞中没有发现分离的F1。FO比F1更难研究，因为它没有膜是不稳定的。另一个限制是通过FlFO合成ATP需要完整膜的相对侧之间的电分离以及产生和维持离子梯度的手段。传统的细胞膜环境提供方法在观察F1 FO的旋转方面效果有限，本课题的目的是开发新的细胞膜环境提供方法，并利用这些方法研究F1 FO合成ATP的机制。我们将开发两种新的技术，用于将大膜蛋白重构成封闭的，能量充足的脂质双层，适用于高数值分辨率的光学显微镜，并探索它们在高分辨率单分子测量中的用途，测量附着在全功能F1 FO和孤立FO上的金纳米颗粒标记的旋转。我们将使用来自大肠杆菌的H+-偶联F1 FO，大肠杆菌是一种模式生物，如果可能的话，还将使用来自鞑靼伊利诺菌和各种其他细菌物种的H+-和Na+-偶联F1 FO。

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.