Roles of Hydration and Lipids in Mechanosensitive Channel Gating

描述(申请人提供)：MSCL是一种原核生物渗透压释放阀，是最被理解的机械敏感通道。作为一个结构易于处理和方便的模型系统，它提供了一个独特的机会来获得包括蛋白质、周围脂质和水的门控的连贯物理图像。在张力的作用下，五聚体蛋白质在膜平面上膨胀约20nm2，打开一个3 nm的水孔。对野生型和功能增益突变体中多个导电态之间的跃迁进行的热力学分析强烈表明，这种跃迁涉及两个门的顺序作用。先前提出的正门虹膜状作用模型在实验上得到了很好的支持。它预测，通道的开放必须与内孔表面的大量水化、蛋白质-脂肪界面的大幅增加以及蛋白质和脂肪双层之间的厚度不匹配有关。对主门亲水性突变的功能分析表明，开放途径中的限速步骤很可能是孔润湿的关键事件。最近，MSCL被发现是冷激活的，有迹象表明，周围双层的扰动改变了温度依赖性的斜率。该提议解决了两个主要问题：气孔水化对浇注的能量学和动力学的贡献是什么，以及边界脂的构象状态是否会赋予高度的温度敏感性？为了解决通道机制的这些重要细节，我们提出：(1)分子动力学模拟孔扩张过程中的润湿/去湿过程，额外的热力学计算蛋白质表面和‘蒸气塞’的界面能；(2)实验膜片钳分析孔的水化性质改变的突变体，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；(4)不同构象的通道蛋白对脂双层的扰动的计算分析。这项研究计划的实施有望阐明疏水门作用的一般原理，以及脂质环境在设置感觉通道的温度依赖性中的作用。

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.