Structural Basis of Chemoreception

描述(申请人提供)：细菌几乎无处不在，在工业和环境中扮演着重要的角色，对人类和其他有机体的健康和疾病都是重要的参与者。它们也是小的、易于操作的模型细胞，可以用于研究基础细胞生物学研究。可移动的细菌，包括许多重要的病原体，不断地监控它们的环境，以便游向营养物质而远离毒素，这一过程被称为趋化性。引诱剂和驱避剂与化学感受器结合，化学感受器通常位于高度合作的有序阵列中聚集在一起的细胞的极点。激活的化学感受器阵列使蛋白质信使磷酸化，而蛋白质信使又与鞭毛马达结合，控制马达逆转的速度，最终控制细胞是继续前进还是改变方向。虽然X射线结晶学和核磁共振光谱等强大的方法已经在近原子分辨率下揭示了某些化学受体的单个结构域的结构，但它们没有揭示化学受体是如何在活细胞内排列的，也没有揭示阵列协同性的结构基础。相反，我们已经开始用一种新兴的技术--电子冷冻断层扫描来解决这些问题，这种技术可以“大分子”(1-5 nm)分辨率对完整的细菌细胞进行三维重建，这足以可视化单个受体二聚体。简而言之，细菌培养物在横跨EM栅格的薄膜中骤然冷冻，然后随着样品围绕一个或两个轴递增倾斜，从一系列角度进行成像。然后从图像中计算三维重建，并对具有共同特征的子区域进行平均以提高信噪比。在最近的工作中，我们证明了细菌化学感受器阵列普遍排列在一个保守的，12 nm的受体二聚体三聚体六聚体晶格中，在这里，我们建议通过成像完全激活和失活的状态来扩展这一工作的分辨率。这应该揭示蛋白质在阵列中是如何排列的，以及激活和阵列协同性的结构基础。这些信息反过来将帮助我们了解细菌如何在健康和疾病中发挥作用，并可能提出新的抗生素目标或策略。

DESCRIPTION (provided by applicant): Bacteria are nearly ubiquitous, play vital roles in industry and the environment, and are important actors in both health and disease for humans and other organisms. They are also small, easily-manipulable model cells that can be used to study basic cell biological studies. Motile bacteria, including many important pathogens, constantly monitor their environment in order to swim towards nutrients and away from toxins, a process called chemotaxis. Attractants and repellents bind to chemoreceptors, which are typically found at the poles of cells grouped together in highly cooperative, ordered arrays. Activated chemoreceptor arrays phosphorylate a protein messenger which in turn binds to flagellar motors, governing the rate of motor reversals and, ultimately, whether the cell continues to move forward or changes direction. While powerful methods like X-ray crystallography and NMR spectrometry have revealed the structures of individual domains of certain chemoreceptors at near-atomic resolution, they have not revealed how the chemoreceptors are arranged inside living cells or the structural basis of array cooperativity. Instead, we have begun to address these issues with an emerging technology, electron cryotomography, which can produce 3-D reconstructions of intact bacterial cells at "macromolecular" (1-5 nm) resolution, which is sufficient to visualize individual receptor dimers. Briefly, bacterial cultures are plunge-frozen in thin films across EM grids and then imaged from a range of angles as the sample is tilted incrementally around one or two axes. 3-D reconstructions are then calculated from the images, and sub-regions with common features can be averaged to increase the signal-to-noise ratio. Following recent work in which we showed that bacterial chemoreceptor arrays are universally arranged in a conserved, 12-nm hexameric lattice of trimers-of-receptor-dimers, here we propose to extend that work in resolution and by imaging fully-activated and -deactivated states. This should reveal how the proteins are arranged within the array as well as the structural basis of activation and array cooperativity. This information will in turn help us understand how bacteria accomplish their roles in health and disease and perhaps suggest new antibiotic targets or strategies.