Structural Basis of Chemoreception

描述（由申请人提供）：细菌几乎无处不在，在工业和环境中发挥着重要作用，是人类和其他生物体健康和疾病的重要因素。它们也是小的，易于操作的模型细胞，可用于研究基本的细胞生物学研究。能动的细菌，包括许多重要的病原体，不断地监测它们的环境，以便游向营养物并远离毒素，这一过程称为趋化性。吸引剂和排斥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的细胞两极。激活的化学感受器阵列磷酸化蛋白质信使，蛋白质信使又与鞭毛马达结合，控制马达逆转的速率，最终控制细胞是继续前进还是改变方向。虽然强大的方法，如X射线晶体学和核磁共振光谱法已经揭示了近原子分辨率的某些化学感受器的单个结构域的结构，但它们并没有揭示化学感受器如何在活细胞内排列或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来解决这些问题，电子冷冻断层扫描，它可以在“大分子”（1-5 nm）分辨率下产生完整细菌细胞的3-D重建，这足以使单个受体二聚体可视化。简单地说，细菌培养物在EM网格上以薄膜形式速冻，然后随着样品围绕一个或两个轴逐渐倾斜，从一系列角度成像。然后从图像计算3-D重建，并且可以对具有共同特征的子区域进行平均以增加信噪比。在最近的工作中，我们表明，细菌化学感受器阵列普遍安排在一个保守的，12纳米的六聚体晶格的三聚体的受体二聚体，在这里，我们建议扩展这项工作的分辨率和成像完全激活和失活状态。这将揭示蛋白质如何在阵列中排列，以及激活和阵列协同性的结构基础。这些信息将反过来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靶点或策略。

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.