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完成的，蛋白质如此之小，以至于10亿个蛋白质可以装进一个句号。许多这些过程都需要蛋白质的集合来组装成功能性的生物机器，其中一些是在细胞膜中发现的。细菌的鞭毛马达就是这样一个机器，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，我们用一个小标记标记它们，当我们用合适颜色的光照射它们时，它会发光，通过测量马达中这种发光的总亮度与单个标签的亮度的关系，我们可以估计出有多少被标记的蛋白质存在。机器的功能状态由电机转速给出。通过一根鞭毛丝将整个细胞绑在一个表面上，观察细胞围绕这一点旋转，或者将细胞贴在一个表面上，检测放置在自由鞭毛丝上的小标签的位置，这两种方法都可以监测到这一点。细菌鞭毛马达是一种巧妙的装置，它跨越细胞膜，将离子的一些电能和化学能转化为马达的旋转机械运动，从而使附着的螺旋细丝旋转，使细胞在液体环境中游动。以前我们发现，马达的单个部件会被迅速更换，这可能意味着生物部件会磨损，需要更换，就像人造机器一样。现在有证据表明，其中一些成分可能是由推动旋转的离子存在而保持在原位的。为了充分测试蛋白质是否真的会降解，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部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，以达到单个蛋白质分子的精度，同时监测马达在各种修饰细菌细胞中的旋转速度，我们可以精确和动态地控制跨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.