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Roles of Hydration and Lipids in Mechanosensitive Channel Gating

水合和脂质在机械敏感通道门控中的作用

基本信息

批准号：
7612737
负责人：
SERGEI I SUKHAREV
金额：
$ 32.44万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-29 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7612737
关键词：
Address Affect Area Attention Behavior Biological Models Charge Cholinergic Receptors Computer Analysis Computer Simulation Crystallography Cysteine Data Dependence Dependency Disulfides Engineering Entropy Environment Equilibrium Event Experimental Models Five-Year Plans Free Energy Funding Histidine Hydration status Hydrogen Bonding Iris Kinetics Lipid Bilayers Lipids Location Measurement Measures Membrane Membrane Proteins Modeling Modification Molecular Conformation Mutation N-terminal Nature Pathway interactions Play Positioning Attribute Process Property Protein Conformation Proteins Radial Relative (related person)Research Personnel Role Sensory Simulate Solvents Structural Models Structure Study models Surface Temperature Testing Thermodynamics Thick Time Vestibule Water Work aqueous base computer studies constriction crosslink design experimental analysis gain of function implementation research interfacial molecular dynamics mutant patch clamp reconstitution research study sensor simulation success tool vapor

项目摘要

DESCRIPTION (provided by applicant): MscL, a prokaryotic osmolyte release valve is the best understood mechanosensitive channel. As a structurally tractable and convenient model system, it offers a unique opportunity to obtain a coherent physical picture of gating that includes the protein, surrounding lipids and water. When forced by tension, the pentameric protein expands in the plane of the membrane by about 20 nm2, opening a 3-nm aqueous pore. Thermodynamic analyses of transitions between multiple conducting states in wild-type and gain-of- function mutants strongly suggest that the transition involves a sequential action of two gates. The previously proposed model of iris-like action of the main gate has been well supported experimentally. It predicts that channel opening must be associated with a massive hydration of the inner pore surface, substantial increase of protein-lipid interface and a thickness mismatch between the protein and the lipid bilayer. Functional analysis of hydrophilic mutations in the main gate suggests that the rate-limiting step in the opening pathway is likely to be the critical event of the pore wetting^ Recently MscL was found to be cold-activated and there are indications that perturbations in the surrounding bilayer change the slope of the temperature dependence. The proposal addresses two main questions: what is the contribution of pore hydration to the energetics and kinetics of gating, and could the conformational state of boundary lipids confer steep temperature sensitivity? To resolve these important details of the channel mechanism, we propose: (1) molecular dynamics simulations of wetting/dewetting processes in the course of pore expansion with additional thermodynamic calculations of interfacial energies for the protein surface and 'vapor plug'; (2) experimental patch-clamp analysis of mutants with altered hydration properties of the pore, including thermodynamic cycles with protonated and de-protonated histidines; (3) experimental studies of MscL temperature dependencies and (4) computational analysis of perturbations of the lipid bilayer by the channel protein in different conformations. Implementation of this research plan promises to clarify the general principles of a hydrophobic gate action and the role of the lipid environment in setting temperature dependencies for sensory channels.

描述（由申请人提供）：MscL，一种原核渗透剂释放阀，是最容易理解的机械敏感通道。作为一个结构上易于处理且方便的模型系统，它提供了一个独特的机会来获得门控的连贯物理图像，包括蛋白质、周围的脂质和水。当受到张力时，五聚体蛋白在膜平面内膨胀约 20 nm2，打开 3 nm 的水孔。对野生型和功能获得突变体中多个导电状态之间转变的热力学分析强烈表明，该转变涉及两个门的连续作用。先前提出的主门虹膜状作用模型已得到实验的良好支持。它预测通道的开放必定与内孔表面的大量水合、蛋白质-脂质界面的大量增加以及蛋白质和脂质双层之间的厚度不匹配有关。对主门中亲水突变的功能分析表明，打开途径中的限速步骤可能是孔润湿的关键事件^最近发现MscL是冷激活的，并且有迹象表明周围双层的扰动改变了温度依赖性的斜率。该提案解决了两个主要问题：孔隙水化对门控能量学和动力学的贡献是什么？边界脂质的构象状态是否可以赋予陡峭的温度敏感性？为了解决通道机制的这些重要细节，我们建议：（1）孔扩张过程中润湿/反润湿过程的分子动力学模拟，并对蛋白质表面和“蒸气塞”的界面能进行额外的热力学计算； (2) 孔水合特性改变的突变体的实验膜片钳分析，包括质子化和去质子化组氨酸的热力学循环； (3) MscL 温度依赖性的实验研究和 (4) 不同构象的通道蛋白对脂质双层的扰动的计算分析。该研究计划的实施有望阐明疏水门作用的一般原理以及脂质环境在设定感觉通道温度依赖性中的作用。