Correlations: Structure-Dynamics-Functions in Channels

描述(申请人提供)：单通道和多通道电导测量，固态核磁共振的高分辨率结构和动态表征，以及高水平的计算工作将导致结构-功能和动态-功能关联。这些将极大地帮助我们理解离子通道的电导、门控和阻断机制，以及众多的功能细节。以前通过这笔赠款提供的支持已经导致了定义功能机制、离子特异性和Granicidin A效率的新关联，以及打开和关闭这一通道的独特模型。对甲型流感病毒的M2 H通道也有了初步的机制认识。全长M2蛋白的电生理和核磁共振研究表明，膜蛋白对双层环境是多么敏感，以及这些结构如何具有动态过程，在水溶性蛋白中很少观察到。 在这里，我们建议刻画M2 H通道中的结构-动力学-功能关联，从而导致机械理解。该蛋白是一个已被证实的药物靶点，也是第一个被克隆和表达的H通道。这种结构的几个关键特征包括在通道阻滞剂存在下可以扭结的单个跨膜螺旋，似乎负责H选择性和酸门控的组氨酸四元体，以及至少在关闭状态下与组氨酸相互作用的色氨酸四元体。高分辨的骨架结构和动力学，作为pH的函数，将获得该蛋白质以及不同条件下组氨酸的pKA。这些努力将以蛋白质的建模和功能特性为指导。 这项工作对于理解膜蛋白的一般功能，以及如何在层状相脂环境中实现和表征离子特异性、电导效率、门控和阻断具有广泛的意义。

DESCRIPTION (provided by applicant): Single and multi-channel conductance measurements, high resolution structural and dynamic characterization by solid-state NMR, and high level computational efforts will lead to structure-function and dynamic-function correlations. These will greatly facilitate our understanding of ion channel conductance, gating and blockage mechanisms, as well as numerous functional details. Previous support through this grant has led to novel correlations for defining functional mechanisms, ion specificity and efficiency in gramicidin A, as well as unique models for opening and closing this channel. Initial mechanistic insights have also been obtained for the M2 H+ channel from influenza A virus. Electrophysiological and NMR studies of the full length M2 protein have illustrated how sensitive membrane proteins are to their bilayer environment and how these structures can have dynamic processes rarely, if ever, observed in water soluble proteins. Here, we propose to characterize structure-dynamic-function correlations in the M2 H+ channel leading to mechanistic understandings. This protein is a proven drug target and it is the first H+ channel to be cloned and expressed. Several key features of this structure include the single transmembrane helix that can be kinked in the presence of a channel blocker, a histidine tetrad that appears to be responsible for H+ selectivity and acid gating, and a tryptophan tetrad that interacts with the histidines, at least in the closed state. High resolution backbone structure and dynamics, as a function of pH, will be obtained for this protein, as well as the pKas of the histidines under various conditions. These efforts will be guided by both modeling and functional characterizations of the protein. This work has broad implications for understanding how, in general, membrane proteins function and how ion specificity, conductance efficiency, gating and blockage can be achieved and characterized all in a lamellar phase lipid environment.