Stoichiometry and turnover of the bacterial flagellar switch protein FliN.
Stoichiometry and turnover of the bacterial flagellar switch protein FliN.
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DOI：
10.1128/mbio.01216-14
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发表时间：
2014-07-01
期刊：
影响因子：
6.4
通讯作者：
Armitage JP
中科院分区：
文献类型：
--
作者：
Delalez NJ;Berry RM;Armitage JP
生物复合物中的一些蛋白质在复合物发挥功能时与游离蛋白质池交换。有证据表明，蛋白质交换可能是适应机制的一部分。细菌鞭毛马达是最复杂的生物机器之一，也是研究大型多聚体复合物中蛋白质动力学的理想模型系统。最近的研究表明，开关复合物中FliM的拷贝数和交换的FliM的分数随着鞭毛旋转的方向而变化。在这里，我们研究了化学计量和营业额的另一个开关复杂的组件，FliN，标记的荧光蛋白CyPet，在大肠杆菌。我们的研究结果证实，在体内，FliM和FliN形成化学计量比为1：4的复合物，并作为一个单元发挥作用。我们估计野生型马达含有120 ± 26个FliN分子。仅顺时针（CW）或逆时针（CCW）旋转的马达分别含有114 ± 17和144 ± 26个FliN分子。CCW-CW FliN拷贝数的比率为1.26，非常接近先前报道的FliM的比率1.29。我们还测量了FliN分子的交换，其具有与FliM相似的时间尺度和对旋转方向的依赖性，与FliM-FliN作为一个单元的交换一致。我们的工作证实了多聚体蛋白质复合物的高度动态性质，并表明在生理条件下，这些机器可能不是平均固定方法所建议的稳定，完整的结构，而是可以响应和适应不断变化的环境的不完整环。 鞭毛是细菌细胞中最复杂的结构之一，其核心马达蛋白在物种间是保守的。现在有证据表明，这些运动蛋白的周转取决于运动活动，这表明周转对功能很重要。开关复合物将化学传感信号传输到转子，并且我们通过使用单细胞测量表明，交换分子的拷贝数和分数都随转子的旋转偏置而变化。当马达被锁定在逆时针旋转时，拷贝数与通过平均、固定方法确定的拷贝数相似，但是当被锁定在顺时针方向时，拷贝数低得多，这表明开关复合环是不完整的。我们的研究结果表明，电机重塑是一个重要的组成部分，在电机调谐响应和适应。
Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments. The flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.