The bacterial mechanosentitive channel as a multimodal sensor device

描述(申请人提供)：识别关键参数的细胞传感器，如脂质双层中的侧压力和细胞质的水合程度，不仅将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生物灵感传感器设备，用于探测环境和筛选潜在的药物。小电导机械敏感通道(MSCs)是一种普遍存在的渗透压释放通道，存在于所有有细胞壁的生物门中。大肠杆菌间充质干细胞是被理解得最好的代表，它被膜张力直接激活，并被细胞质内聚合物增加的拥挤压力抑制。晶体结构预测MSCs的跨膜区感受张力，而空心胞浆域(CAGE)感知拥挤压力，并根据细胞质水合程度调节张力敏感性和开放时间。此外，由于门相对于膜中面的位置不对称，MSCs对内叶中的侧向压力/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，并探讨侧向压力传感器的实用设计。更具体地说，我们建议(1)模拟几种生物活性化合物插入到脂质双层中，并使用分子动力学计算侧向压力分布的变化。然后，我们将使用这些结果来模拟MSCs的扩张，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，我们将对MSCs进行重新设计，以提高敏感性和稳定性。该通道将在存在引起已知压力变化的物质的情况下进行校准，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。为了了解胞质拥挤灭活间充质干细胞的机制，我们将(3)计算探讨空笼结构域的构象动力学、不同构象中的排斥体积和可压缩性，以及与孔衬里螺旋的耦合。(4)通过计算确定具有重要功能的构象将通过突变和详细的膜片钳分析在拥挤剂存在的情况下进行实验测试。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。它还将揭示间充质干细胞膜包埋区和细胞质结构域之间的变构相互作用，以及细胞测量细胞质水合程度的生物物理原理，从而为设计生物启发的嗅觉传感器打开机会。

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.