Proton Conduction Pathways in Proton Channel Proteins

项目摘要： 质子通道蛋白增强质子穿过细胞膜的流动，并进化出精细的控制 质子选择性和传导性，以有效地实现其功能，同时保持细胞的完整性。 通过形成动态质子传导途径，模拟在稀酸中观察到的水线 对于质子扩散，质子快速且选择性地沿由以下组成的氢键网络沿着移动 封闭的水和可电离的侧链分散在质子通道蛋白的内腔中。单程质子 通道介导质子传导性是通过导水丝，导水丝是稳定的内腔沃茨组织 由极性蛋白质组成。导水丝是在高分辨率晶体中观察到的，因此得到了很好的研究 结构，但无论它们是移动的还是静态的，以及它们的动态如何影响质子传导性， 不清楚调节质子选择性和传导性的另一种方法是通过瞬态水线， 被认为是形成和消散，以允许质子通量通过良好包装的非极性部分。当瞬态水 在分子动力学（MD）模拟中假设了金属丝，但它们从根本上难以测试 实验性的最后，质子通道也使用质子化和去质子化的质子穿梭机制 通过可电离的侧链，如His，Glu和Asp，来调节质子电导，但目前还不清楚 这些侧链介导孔溶剂化的程度，以及质子穿梭机制是否导致净转运 水。这项工作将解决质子通道蛋白介导质子通量的这些机制： (1)表面上稳定的氢键网络的导水线和蛋白质极性基团，（2）瞬态 水导线，和（3）质子梭组成的电离侧链。 通过我们对天然质子通道，甲型流感病毒基质蛋白2（M2）的研究， 设计的质子通道，我们将测试的假设，（1）引导和瞬态水线内质子 通道蛋白赋予它们的选择性并决定它们传导质子的能力，以及（2）质子穿梭是 不仅在确定这些质子通道的传导率方面是必要的，而且在以下方面也起着关键作用： 调节质子和水的渗透性。在目标1中，我们将检查导水管是否是移动的， 通过多维红外光谱对M2质子通道和疾病相关突变体进行静态分析。我们 在存在和不存在药物的情况下进行测量将使我们能够确定这些药物的动力学如何变化。 网络影响质子传导，以及它们如何随着药物结合和耐药突变而变化， 这对确定新的抗病毒策略至关重要。在目标2中，我们通过以下方式测试瞬态水线的假设： 具有不同长度非极性区域的新型质子通道的从头设计和表征。在 R 00相（目标3），我们研究了可电离侧链如何增强孔隙水化，并研究了 质子化/去质子化事件导致质子和水的共易位。

PROJECT ABSTRACT: Proton channel proteins potentiate the flow of protons across cell membranes, and have evolved fine control over proton selectivity and conductivity to efficiently achieve their function, while maintaining cellular integrity. Through formation of dynamic proton conduction pathways which mimic the water wires observed in dilute acid for proton diffusion, protons move rapidly and selectively along a hydrogen-bonding network composed of confined water and ionizable sidechains scattered within the lumen of proton channel proteins. One way proton channels mediate proton conductivity is through guide water wires, which are stable lumenal waters organized by polar protein groups. Guide water wires are well-studied as they are observed in high-resolution crystal structures, but whether they are mobile or static and how their dynamics affects proton conductivity remains unclear. Another way to modulate proton selectivity and conductivity is through transient water wires, which are thought to form and dissipate to allow for proton flux through well-packed apolar segments. While transient water wires have been hypothesized in molecular dynamics (MD) simulations, they are fundamentally difficult to test experimentally. Finally, proton channels also use proton shuttle mechanisms of protonation and deprotonation through an ionizable sidechain, such as His, Glu, and Asp, to tune proton conductance, but it is unclear the extent these sidechains mediate pore solvation, and whether the proton shuttle mechanism leads to a net transit of water. This work will address these mechanisms by which proton channel proteins mediate proton flux: the (1) seemingly stable hydrogen-bonding networks of guide water wires and protein polar groups, (2) transient water wires, and (3) proton shuttles composed of ionizable sidechains. Through our proposed study of a natural proton channel, the influenza A matrix protein 2 (M2), and de novo designed proton channels, we will test the hypotheses that (1) guide and transient water wires within proton channel proteins confer their selectivity and dictate their capacity to conduct protons, and (2) proton shuttles are not only necessary in defining the conduction rates of these proton channels, but also play critical roles in modulating proton and water permeability. In Aim 1, we will examine whether guide water wires are mobile or static by multidimensional infrared spectroscopy on M2 proton channels and the disease-relative mutants. Our measurements in the presence and absence of drugs will allow us to determine how the dynamics of these networks affect proton conductance, and how they change with drug binding and resistance mutations, which is critical to identifying new antiviral strategies. In Aim 2, we test the hypothesis of transient water wires through the de novo design and characterization of novel proton channels with varying lengths of apolar regions. In the R00 phase (Aim 3), we examine how ionizable sidechains potentiate pore hydration and investigate whether protonation/deprotonation events lead to the cotranslocation of protons and water.