The bacterial mechanosentitive channel as a multimodal sensor device

作为多模式传感器装置的细菌机械感应通道

• 批准号: 8471474
• 负责人: SERGEI I SUKHAREV
• 金额: $ 32.89万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471474
• 关键词: Affinity; Anti-Bacterial Agents; Antibiotics; Biomedical Engineering; Cell Wall; Cell membrane; Cell physiology; Cells; Chemistry; Ciprofloxacin; Coupling; Crowding; Cytoplasm; Cytoplasmic Tail; Data; Development; Devices; Engineering; Environment; Hydration status; Inner Leaflet of the Lipid Bilayer; Knowledge; Lateral; Lipid Bilayers; Lipids; Measurement; Measures; Mechanics; Membrane; Modality; Modeling; Molecular; Molecular Conformation; Monitor; Mutagenesis; Mutation; Nature; Organism; Outcome Study; Parabens; Pathway interactions; Penetration; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Polymers; Positioning Attribute; Property; Proteins; Relative (related person); Resistance; Simulate; Structure; Surface; Techniques; Testing; Tetracyclines; Transducers; Transmembrane Domain; Vestibule; analog; base; density; design; drug development; drug testing; in vivo; innovation; intercalation; interfacial; intermolecular interaction; macromolecule; molecular dynamics; patch clamp; pressure; prototype; public health relevance; research study; response; screening; sensor

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.

描述（由申请人提供）：识别用于关键参数（例如脂质双层中的侧向压力和细胞质水合程度）的细胞传感器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用于探测环境和筛选潜在药物的生物启发传感器装置。机械敏感小电导通道（MscS）是一种普遍存在于所有具有细胞壁的生物门中的渗透剂释放通道。大肠杆菌MscS，最好的理解的代表，是直接激活的膜张力和抑制细胞质中的聚合物的拥挤压力增加。晶体结构预测，跨膜结构域的MscS的感觉张力，而中空的胞质结构域（笼）感知拥挤的压力和调整张力的敏感性和开放的持续时间根据细胞质水合的程度。此外，由于门相对于膜中平面的不对称位置，MscS对内瓣叶中的侧向压力/张力更敏感。两亲性物质强烈影响激活张力，因此该通道可用作药物分配到天然细菌膜中的内源性传感器。在这个项目中，我们联合收割机的实验和计算的努力，旨在探索不同的传感模式的MSCS和方法的实际设计的侧压力传感器。更具体地说，我们建议（1）模拟嵌入的几种生物活性化合物到脂质双层和计算的变化，在横向压力分布使用分子动力学。然后，我们将使用这些结果来模拟MscS扩展，以确定在通道中的分子间相互作用，可以影响不对称张力的敏感性。(2)基于这些结果，我们将重新设计MscS，以提高灵敏度和稳定性。该通道将在存在导致使用独立的表面化学技术确定的已知压力变化的物质的情况下进行校准，然后用于几种抗生素及其合成类似物的实际筛选和表征。为了理解细胞质拥挤导致MscS失活的机制，我们将（3）计算探索中空笼结构域的构象动力学，不同构象中的排斥体积和压缩，以及与孔衬螺旋的耦合。(4)通过计算确定为功能重要的构象将通过诱变和在拥挤剂存在下的详细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，用于监测两亲物质通过天然细菌膜的掺入和渗透。它还将揭示MscS的膜嵌入结构域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，从而为生物启发的生物传感器的设计提供了机会。