Stoichiometry and turnover in single, functioning membrane protein complexes
Stoichiometry and turnover in single, functioning membrane protein complexes
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DOI：
10.1038/nature05135
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发表时间：
2006-09-21
期刊：
影响因子：
64.8
通讯作者：
Armitage, Judith P.
中科院分区：
文献类型：
--
作者：
Leake, Mark C.;Chandler, Jennifer H.;Armitage, Judith P.
许多基本的细胞过程是由位于细胞膜中的复杂生物机器进行的。细菌鞭毛马达是一种大的跨膜蛋白复合物，其作为离子驱动的旋转马达来推动细胞通过液体培养基（1-3）。在电机内，MotB是定子的一个部件，其将离子流耦合到扭矩产生并将定子锚定到单元壁（4，5）。在这里，我们已经调查了蛋白质的化学计量，动力学和营业额的MotB与单分子精度在大肠杆菌中的细菌鞭毛马达功能。我们通过旋转拴系的细胞体（6）来监测运动功能，并通过全内反射荧光显微镜同时测量运动中用绿色荧光蛋白（GFP-MotB）标记的MotB分子的数量和动力学.通过单个GFP分子的逐步光漂白计数荧光团显示每个马达含有类似于22个拷贝的GFP-MotB，与类似于11个定子（每个定子含有两个MotB分子）一致。我们还观察到一个膜池，大约有200个GFP-MotB分子以大约0.008 μ m（2）s（-1）的速度扩散.光漂白后的荧光恢复和光漂白中的荧光损失表明，GFP MotB在膜池和马达之间的周转率为0.04 s（-1）：马达中给定定子的停留时间仅为0.5 min。这是第一次直接测量功能分子机器内蛋白质亚基的数量和快速周转。
Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media(1-3). Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall(4,5). Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body(6), and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP - MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains similar to 22 copies of GFP - MotB, consistent with similar to 11 stators each containing two MotB molecules. We also observed a membrane pool of similar to 200 GFP - MotB molecules diffusing at similar to 0.008 mu m(2) s(-1). Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP MotB between the membrane pool and motor with a rate constant of the order of 0.04 s(-1): the dwell time of a given stator in the motor is only similar to 0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.