The bacterial mechanosentitive channel as a multimodal sensor device

描述（由申请人提供）：识别关键参数的细胞传感器，如脂质双分子层的侧压和细胞质水化程度，不仅可以促进我们对细胞生理学和力学的基本理解，而且还可以用于开发用于探测环境和筛选潜在药物的仿生传感器设备。小电导机械敏感通道（Mechanosensitive Channel of Small conductivity, MscS）是一种普遍存在于所有具有细胞壁的生物门中的渗透物释放通道。大肠杆菌间充质干细胞是最好的代表，它被膜张力直接激活，并被细胞质中聚合物拥挤压力的增加所抑制。晶体结构预测MscS的跨膜结构域感知张力，而中空的细胞质结构域（笼）感知拥挤压力，并根据细胞质水化程度调节张力敏感性和开放时间。此外，由于门相对于膜中层的不对称位置，MscS对内部小叶的侧压力/张力更敏感。激活张力受到两亲性物质的强烈影响，因此该通道可以作为药物分配到天然细菌膜的内源性传感器。在这个项目中，我们结合了三个小组的实验和计算工作，旨在探索MscS的不同传感模式，并接近侧向压力传感器的实际设计。更具体地说，我们建议(1)模拟几种生物活性化合物嵌入脂质双分子层，并使用分子动力学计算侧压力分布的变化。然后，我们将使用这些结果来模拟MscS的扩展，以确定通道中可能影响对不对称张力敏感性的分子间相互作用。(2)基于这些结果，我们将重新设计MscS以获得更高的灵敏度和稳定性。该通道将在存在导致已知压力变化的物质的情况下进行校准，这些物质使用独立的表面化学技术确定，然后用于几种抗生素及其合成类似物的实际筛选和表征。为了了解细胞质拥挤导致MscS失活的机制，我们将(3)通过计算探索中空笼结构域的构象动力学，排除不同构象的体积和可压缩性，以及与孔衬螺旋的耦合。(4)通过计算确定的具有重要功能的构象将在拥挤因子存在的情况下通过诱变和详细的膜片钳分析进行实验测试。该项目将建立第一个基于传感器的平台，用于监测两亲性物质通过天然细菌膜的结合和渗透。它还将揭示MscS的膜嵌入域和细胞质域之间的变构相互作用，以及细胞测量细胞质水化程度的生物物理原理，从而为设计生物启发渗透传感器提供机会。

DESCRIPTION (provided by applicant): Identification of cellular sensors for key parameters such as lateral pressure in the lipid bilayer and degree of cytoplasm hydration will not only advance our basic understanding of cell physiology and mechanics, but can also be used in the development of bio-inspired sensor devices for probing the environment and for screening potential pharmaceuticals. Mechanosensitive Channel of Small Conductance (MscS) is a ubiquitous osmolyte release channel found in all phyla of organisms with cell walls. Esherichia coli MscS, the best understood representative, is directly activated by membrane tension and inhibited by increased crowding pressure of polymers in the cytoplasm. Crystal structures predict that the transmembrane domain of MscS senses tension, whereas the hollow cytoplasmic domain (cage) perceives crowding pressure and adjusts tension sensitivity and duration of opening according to the degree of cytoplasmic hydration. Additionally, due to the asymmetric position of the gate relative to the membrane midplane, MscS is more sensitive to lateral pressure/tension in the inner leaflet. Activating tensions are strongly influenced by amphipathic substances, and therefore the channel can be used as an endogenous sensor of drug partitioning into the native bacterial membrane. In this project we combine experimental and computational efforts of three groups aimed to explore different sensing modalities of MscS and approach the practical design of a lateral pressure sensor. More specifically, we propose to (1) simulate intercalation of several biologically active compounds into the lipid bilayer and compute changes in lateral pressure profiles using Molecular Dynamics. We will then use these results to simulate MscS expansion to identify intermolecular interactions in the channel that can influence sensitivity to asymmetric tension. (2) Based on these results, we will re-engineer MscS for higher sensitivity and stability. The channel will be calibrated in the presence of substances causing known pressure shifts determined using independent surface chemistry techniques, and then used for practical screening and characterization of several antibiotics and their synthetic analogs. In order to understand the mechanism of MscS inactivation by cytoplasmic crowding, we will (3) computationally explore the conformational dynamics of the hollow cage domain, excluded volumes and compressibilities in different conformations, and the coupling with the pore-lining helices. (4) Conformations identified by computations as functionally important will be tested experimentally through mutagenesis and detailed patch-clamp analysis in the presence of crowding agents. The project will establish the very first sensor-based platform for monitoring incorporation and permeation of amphipathic substances through native bacterial membranes. It will also reveal the allosteric interplay between the membrane-embedded and cytoplasmic domains of MscS, and the biophysical principle by which cells measure the extent of cytoplasmic hydration, thus opening the opportunity for the design of bio-inspired osmosensors.