Understanding protein interaction and turnover in the bacterial flagellar motor

了解细菌鞭毛运动中的蛋白质相互作用和周转

• 批准号: BB/M008657/1
• 负责人: Judith Armitage
• 金额: $ 42.83万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM008657%2F1
• 关键词: Understanding; protein; interaction; turnover; bacterial

The majority of bacterial species can move, many swimming. They use swimming to reach their best environment for growth and survival. For pathogenic bacteria that can be a wound, or the lung or gut wall, for beneficial bacteria it could be a root, while for many bacteria it might be a surface where they can grow as a slime protected colony. Swimming uses a mechanism unique to bacteria, making it a possible site for controlling bacterial activity such as colonisation. However, to control swimming behaviour, and thus colonisation, we need to know how swimming works. Swimming relies on the rotation of rigid, helical flagellar filaments, the diameter of just over a hundredth of a millimetre. It was initially thought the bacterial flagellar filament was rotated by a structure in the bacterial membrane that operated like a tiny, stable electric motor with a ring of proteins, rotor proteins, anchored to the helical filament, which rotate against a fixed ring of proteins (stators) anchored to the cell wall. As charged particles move through the stator proteins a change in local charge interactions between the stator and rotor proteins drives the rotor ring round, at speeds of up to 1300 rev per sec. While this general mechanism is correct, a few years ago we showed that the proteins within a rotating motor are not in fixed rings, but some of the proteins within the rings are swapping with free proteins even though the motor is still spinning-rather like changing rotating parts of a car engine while driving at full speed down the motorway. Indeed more recently we found that if you removed the driving force, the ion gradient, altogether the supposedly anchored ring of stator proteins simply diffuses away from the rotor into the bacterial membrane, returning to the rotor one at a time only when the driving force is reinstated. The motor can also "feel" outside forces on the motor and adds additional components as the external force increases, as happens when the environment gets more viscous, as in a mucous gut or lung wall or when encountering a surface, allowing rotation to continue. The bacterial motor is therefore continually remodelling to allow swimming under a range of conditions. It has been known for a while that unrelated proteins that change concentration or activity during different growth conditions can alter swimming behaviour. It now seems likely that while remodelling these other proteins can "slip" in to the motor and interact with motor proteins to either slow or stabilise the motor structure and alter activity. In this study we intend to (1) characterise the effect on motor structure of these non-motor proteins, (2) to identify the changes that occur in the motor structure when it rotates under different conditions and (3) compare these changes across very different species with motors showing a common core, but with different rotational behaviours. This will allow the development of a more complete understanding of the dynamics of motor proteins and protein:protein interaction that could lead to the design of molecules that will interfer with that rotation and prevent colonisation.

大多数细菌种类可以移动，许多是游泳。它们利用游泳来达到最佳的生长和生存环境。对于致病菌，可以是伤口，或肺或肠壁，对于有益细菌，它可能是根，而对于许多细菌，它可能是一个表面，在那里它们可以作为粘液保护的菌落生长。游泳使用细菌特有的机制，使其成为控制细菌活动（如定植）的可能场所。然而，为了控制游泳行为，从而控制殖民，我们需要知道游泳是如何工作的。游泳依赖于刚性螺旋状鞭毛丝的旋转，直径略大于百分之一毫米。最初认为细菌鞭毛丝是由细菌膜中的一种结构旋转的，这种结构就像一个微小的、稳定的电动机，它有一个蛋白质环，转子蛋白质，锚定在螺旋丝上，它旋转着一个固定在细胞壁上的蛋白质环（定子）。当带电粒子移动通过定子蛋白质时，定子蛋白质和转子蛋白质之间的局部电荷相互作用的变化驱动转子环旋转，速度高达每秒1300转。虽然这种一般机制是正确的，但几年前我们发现，旋转马达中的蛋白质并不在固定的环中，但即使马达仍在旋转，环中的一些蛋白质也会与游离蛋白质交换--这就像在高速公路上全速行驶时更换汽车发动机的旋转部件一样。事实上，最近我们发现，如果你去除驱动力，离子梯度，整个定子蛋白质的所谓锚定环只是从转子扩散到细菌膜中，只有当驱动力恢复时，才一次一个地回到转子。马达还可以“感觉”作用在马达上的外力，并随着外力的增加而增加额外的组件，如当环境变得更加粘稠时，如在粘液肠或肺壁中或当遇到表面时，允许旋转继续。因此，细菌马达不断重塑，以允许在一系列条件下游泳。一段时间以来，人们已经知道，在不同生长条件下改变浓度或活性的不相关蛋白质可以改变游泳行为。现在看来，在重塑的同时，这些其他蛋白质可能会“滑”进马达，并与马达蛋白相互作用，以减缓或稳定马达结构并改变活性。在这项研究中，我们打算（1）研究这些非马达蛋白对马达结构的影响，（2）确定马达结构在不同条件下旋转时发生的变化，（3）比较这些变化在非常不同的物种中，马达显示出共同的核心，但具有不同的旋转行为。这将使我们能够更全面地了解马达蛋白和蛋白质的动力学：蛋白质相互作用，从而设计出能够干扰旋转并防止殖民化的分子。

项目成果

Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

• DOI: 10.1371/journal.pbio.1002039
• 发表时间: 2015-01
• 期刊: PLoS biology
• 影响因子: 9.8
• 作者: Diepold A;Kudryashev M;Delalez NJ;Berry RM;Armitage JP
• 通讯作者: Armitage JP

Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.

• DOI: 10.1098/rstb.2015.0492
• 发表时间: 2016-11-05
• 期刊: Philosophical transactions of the Royal Society of London. Series B, Biological sciences
• 作者: Di Paolo D;Afanzar O;Armitage JP;Berry RM
• 通讯作者: Berry RM