Torque generation in the bacterial flagellar motor

该项目的目的是了解细菌鞭毛马达的机制，这是一种直径约为50 nm（1/20，000毫米）的旋转分子电动马达，最大速度超过100，000 rpm。螺旋桨是约20 nm厚的螺旋鞭毛丝，马达由离子（H+或Na+）沿称为质子动力（PMF）或钠动力（SMF）的电化学梯度流动驱动。这些梯度由跨细胞膜的电压和浓度差组成，是生物能量的主要形式。每个马达的最大功率输出约为百万分之一瓦，比其他已知的分子马达高出约100倍，这些分子马达由细胞的通用能量货币分子ATP提供动力。转子是细胞质膜中的一组环，直径约为45 nm，并由约12个独立的扭矩产生单元包围，这些扭矩产生单元锚定在细胞壁上，并在离子流过时推动转子。我们将使用一系列的生物物理技术，一些相对完善的和其他全新的，以衡量旋转的单一鞭毛电机远远优于以往任何时候都可能在过去。马达太小，与周围的一切太相似，无法在光学显微镜下看到。电子显微镜只对冷冻的样品起作用，所以当马达工作时，电子显微镜对观察马达没有好处。我们通过将微小的金颗粒附着在粘附在细胞外的马达上来解决这个问题，金颗粒会散射大量的光，这样我们就可以看到它们。然后，我们通过拍摄高速视频（高达每秒109，500帧！），或者通过将相同的图像投影到快速位置敏感检测器上。我们将建立在我们实验室最近的实验创新的基础上，这使得有史以来第一次检测到鞭毛马达中的基本扭矩产生步骤。当通过降低Na+浓度来降低SMF时，在仅包含一个单元的电机中观察到14度的步进。扭矩产生单元是包含来自不同物种的成分的嵌合体，使我们能够使用E.大肠杆菌（通常具有H+驱动的马达）。使用Na+驱动的马达确保我们可以让它们慢下来，足以用我们的旧显微镜看到台阶，这比我们刚刚开发的新显微镜慢50倍。我们仍在改进新的显微镜，我们希望它是如此之好，我们可以测量步骤在H+驱动电机没有做任何不寻常的减速电机。我们认为，每一步可能对应于一个或两个离子穿过电机，但需要在一系列不同条件下对许多步进行详细和系统的测量，以确保情况确实如此。为了确保我们正确地解释我们所看到的，我们将开发和使用先进的统计工具，以及电机机制的数学模型。到目前为止，我们只能在改变驱动力的同时观察发动机的旋转-如果你喜欢的话，可以说是“燃料”。我们真的希望能够保持它不动，看看它能推得多厉害。我们将用微小的钴磁铁代替金颗粒作为手柄。我们可以用磁场扭转这些，看看马达如何反应。这将允许对马达进行各种新的探测，因为我们以不同的角度保持它不动，向后推它，让它以不同的速度向前运行。了解鞭毛马达将有助于更广泛的分子马达领域，并将奠定可能的技术应用的基础。它还将有助于实现在分子尺度上设计和建造人造机器的长期目标。

The aim of the project is to understand the mechanism of the bacterial flagellar motor, a rotary molecular electric motor with a diameter of ~50 nm ( 1/20,000th of a mm) and a maximum speed in excess of 100,000 r.p.m. Many species of bacteria navigate their environment by swimming. The propellers are helical flagellar filaments ~20 nm thick and the motor is driven by the flow of ions, either H+ or Na+, down an electrochemical gradient called the protonmotive force (PMF) or sodium-motive force (SMF). These gradients consist of a voltage and a concentration difference across the cell membrane and are the primary form of biological energy. Each motor has a maximum power output of about one million-billionth of a Watt, about 100 times higher than other known molecular motors which are powered by the universal energy currency molecule of the cell, ATP. The rotor is a set of rings in the cytoplasmic membrane, about 45 nm in diameter and surrounded by about a dozen independent torque generating units which are anchored to the cell wall and push on the rotor when ions flow through. We will use a range of biophysical techniques, some relatively well established and others brand-new, to measure the rotation of single flagellar motors far better than has ever been possible in the past. The motor is too small and too similar to everything else that surrounds it to see in a light microscope. Electron microscopes only work on frozen samples, so are no good for seeing the motors when they are working. We get around this by attaching tiny gold particles, which scatter a lot of light so that we can see them, to the bits of the motor that stick outside the cell. We then measure their rotation either by taking high-speed videos (up to 109,500 frames per second!), or by projecting the same image onto a fast position-sensitive detector. We will build on recent experimental innovations in our lab which allowed the first ever detection of the fundamental torque-generating step in the flagellar motor. Steps of 14 degrees were seen in motors containing only one unit, when the SMF was reduced by lowering the Na+ concentration. The torque-generating units were chimeras containing components from different species, allowing us to study a Na+-driven motor with all the genetic tools that are possible using E. coli (which normally has H+-driven motors). Using Na+-driven motors made sure that we could slow them down enough to see the steps with our old microscope, which was 50 times slower than the new one we have just developed. We are still improving the new microscope, and we hope it will be so good that we can measure steps in the H+-driven motor without doing anything unusual to slow the motor down. We believe that each step may correspond to one or two ions crossing the motor, but will need to make detailed and systematic measurements of many steps under a range of different conditions to be sure that this is the case. To make sure we are interpreting what we see correctly, we will develop and use advanced statistical tools, and mathematical models of the motor mechanism. So far, we can only watch the motor spin while varying the driving force - the 'fuel' if you like. We'd really also like to be able to hold it still, and see how hard it can push. We will do this with tiny cobalt magnets replacing the gold particles as handles. We can twist these with a magnetic field, and see how the motor responds. This will allow all sorts of new probes of the motor, as we hold it still at different angles, push it backwards, and let it run forwards at different speeds. Understanding the flagellar motor will contribute to the wider field of molecular motors and will lay the foundations for possible technological applications. It will also contribute towards the long-term goal of designing and building artificial machines at the molecular scale.