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

• 批准号: 7236226
• 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
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-29 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7236226
• 关键词: Address; Affect; Area; Attention; Behavior; Biological Models; Charge; Cholinergic Receptors; Closure; Computer Analysis; Computer Simulation; Constriction procedure; Crystallography; Cysteine; Data; Dependence; Dependency; Disulfides; Engineering; Entropy; Environment; Equilibrium; Event; Experimental Models; Five-Year Plans; Free Energy; Funding; Histidine; Hydration status; Iris; Kinetics; Lipid Bilayers; Lipids; Location; Measurement; Measures; Membrane; Membrane Proteins; Modeling; Modification; Molecular Conformation; Mutation; N-terminal; Nature; Object Attachment; Other Working Groups; Pathway interactions; Personal Satisfaction; Play; Positioning Attribute; Process; Property; Protein Conformation; Proteins; Range; Rate; Relative (related person); Research Personnel; Role; Sensory; Simulate; Solvents; Structural Models; Structure; Study models; Surface; Temperature; Testing; Thermodynamics; Thick; Time; Vestibule; Water; aqueous; base; computer studies; crosslink; design; experimental analysis; gain of function; implementation research; interfacial; molecular dynamics; mutant; patch clamp; radius bone structure; reconstitution; research study; sensor; simulation; success; tool; vapor

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，原核生物渗透释放阀是最佳理解的机械敏感通道。作为一种在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，可以获得包括蛋白质，周围脂质和水在内的门控的连贯的物理图片。当张力强迫时，五聚蛋白在膜的平面上膨胀约20 nm2，打开一个3 nm的水孔。野生型和功能增益突变体中多个导电状态之间过渡的热力学分析强烈表明该过渡涉及两个门的顺序作用。先前提出的主门类似虹膜样作用的模型已得到了实验良好的支持。它预测，通道开口必须与内部孔隙表面的大量水合有关，蛋白质脂质界面的大幅增加以及蛋白质和脂质双层之间的厚度不匹配。主门中亲水性突变的功能分析表明，开场途径中的限制步骤可能是孔润湿的关键事件^最近MSCL被冷激活，并且有迹象表明周围双层的扰动会改变温度依赖性的斜率。该提案解决了两个主要问题：孔隙水合对门控能量和动力学的贡献是什么，边界脂质的构象状态是否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，我们提出：（1）在孔扩展过程中润湿/脱水过程的分子动力学模拟，并通过对蛋白质表面和“蒸气塞”的界面能量进行其他热力学计算； （2）对孔的水合特性改变的突变体的实验性贴片钳分析，包括具有质子化和去骨的组氨酸的热力学循环； （3）MSCL温度依赖性的实验研究和（4）通过不同构象的通道蛋白对脂质双层扰动的计算分析。该研究计划的实施有望阐明疏水栅极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方面的作用。