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，鞭毛马达是一种直径约50纳米（1/20,000毫米）的旋转分子电动机，最大速度超过100,000转/分。许多细菌通过游泳来导航它们的环境。螺旋桨是约20nm厚的螺旋鞭毛细丝，电机由离子流驱动，H+或Na+沿着称为质子动力（PMF）或钠动力（SMF）的电化学梯度向下流动。这些梯度由细胞膜上的电压和浓度差异组成，是生物能量的主要形式。每个马达的最大输出功率约为十亿分之一瓦特，比其他已知的分子马达高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。转子是细胞质膜上的一组环，直径约45纳米，周围有大约12个独立的扭矩产生单元，这些扭矩产生单元固定在细胞壁上，当离子流过转子时，就会对转子施加推力。我们将使用一系列生物物理技术，其中一些相对完善，另一些则是全新的，来测量单个鞭毛马达的旋转，这比以往任何时候都要好得多。马达太小了，与周围的一切都太相似了，无法在光学显微镜下看到。电子显微镜只能在冷冻的样品上工作，所以不能很好地看到马达工作时的样子。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，这些金颗粒会散射很多光，这样我们就能看到它们。然后，我们通过拍摄高速视频（高达每秒109,500帧！）或将相同的图像投射到快速位置敏感探测器上来测量它们的旋转。我们将以实验室最近的实验创新为基础，首次检测到鞭毛马达中产生扭矩的基本步骤。当通过降低Na+浓度来减少SMF时，在只含有一个单元的电机中可以看到14度的步长。产生扭矩的单元是包含不同物种成分的嵌合体，这使我们能够利用大肠杆菌（通常有H+驱动的马达）的所有可能的遗传工具来研究Na+驱动的马达。使用Na+驱动的马达确保我们可以减慢它们的速度，以便用我们的旧显微镜看到台阶，旧显微镜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，我们希望它能非常好，以至于我们可以测量氢离子驱动电机的步长，而不需要做任何不寻常的事情来减慢电机的速度。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骤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，以及运动机制的数学模型。到目前为止，我们只能看着马达旋转，同时改变驱动力——如果你喜欢，也可以称之为“燃料”。我们也希望能够让它保持静止，看看它能有多大的推力。我们将用微小的钴磁铁代替金颗粒作为手柄。我们可以用磁场扭曲它们，看看马达的反应。这将允许对马达进行各种各样的新探测，因为我们以不同的角度保持它静止，向后推它，并让它以不同的速度向前运行。对鞭毛马达的理解将有助于分子马达领域的发展，并为可能的技术应用奠定基础。它还将有助于实现在分子尺度上设计和制造人工机器的长期目标。

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.