Protein turnover studies using single-molecule microscopy in functional bacterial flagellar motors of live cells to assess molecular complex stability

活细胞中的大多数重要活动都是由蛋白质进行的，这些蛋白质非常小，可以容纳10亿个句号。许多这些过程需要蛋白质集合在一起组装成功能性生物机器，其中一些在细胞膜中发现。细菌的鞭毛马达就是这样一台机器，并且是一个理想的研究例子，因为我们已经开发了几种技术，使我们能够观察单个蛋白质并监测机器的功能状态。我们可以看到蛋白质在细胞中的位置，方法是用一个小标记物标记它们，当我们用正确颜色的光照射它时，它会发光，通过测量马达中这种发光的总亮度相对于单个标记物的亮度，我们可以估计有多少标记的蛋白质存在。机器的功能状态由电机速度给出。这可以通过将整个细胞通过其鞭毛丝之一拴在表面上并观察细胞围绕该点旋转来监测，或者通过将细胞贴在表面上并检测放置在游离鞭毛丝上的小标签的位置来监测。细菌鞭毛马达是一种巧妙的装置，它跨越细胞膜并将离子的一些电能和化学能转化为马达的旋转机械运动，从而使附着的螺旋丝旋转并允许细胞在其液体环境中游泳。以前我们发现，马达的单个组件会迅速更换，这可能意味着生物组件会磨损，需要更换，就像它们在人造机器中一样。现在有证据表明，这些成分中的一些可能是由于为旋转提供燃料的离子的存在而保持在适当的位置。为了充分测试蛋白质是否真的降解，周围离子的存在如何影响机器的稳定性，以及机器的功能如何影响零件更换的方式和时间，我们建议监测几个马达组件的运动和数量，精确到单个蛋白质分子，同时监测各种修饰细菌细胞中马达的旋转速度，并动态地控制穿过细胞膜的离子梯度的大小。

Most of the vital activities in living cells are carried out by proteins, so small that 1 billion could fit on a full-stop. Many of these processes require collections of proteins to assemble together into functional biological machines, several of which are to be found in the cell membrane. The flagellar motor of bacteria is such a machine, and is an ideal example to study since we have developed several techniques which allow us to observe the individual proteins as well as monitoring the machine's functional state. We can see where the proteins are located in the cell by tagging them with a small marker which glows when we shine the right colour of light on it, and by measuring the total brightness of this glow in the motor relative to the brightness of just a single tag we can estimate how many of the tagged proteins are present. The functional state of the machine is given by the motor speed. This can be monitored both by tethering the whole cell to a surface via one of its flagellar filaments and watching the cell rotate about this point, or by sticking the cell down to a surface and detecting the position of a small tag placed on a free flagellar filament. The bacterial flagellar motor is an ingenious device which spans the cell membrane and converts some of the electrical and chemical energy of ions into rotary mechanical motion of the motor, thereby bringing about rotation of an attached helical filament and allowing the cell to swim through its liquid surroundings. Previously we discovered that individual components of the motor are rapidly replaced, which may mean that biological components wear out and need replacing just as they do in man-made machines. There is now evidence that the some of these components may be held in place by the presence of the very ions which fuel the rotation. To fully test whether proteins do indeed degrade, how the presence of the surrounding ions affects the stability of the machine and how functioning of the machine affects how and when parts are replaced, we propose to monitor the movement and number of several motor components to a precision of single protein molecules whilst simultaneously monitoring the rotation speed of the motor in varieties of modified bacterial cells in which we can precisely and dynamically control the magnitude of the ion gradient across the cell membrane.