Proton Conduction Pathways in Proton Channel Proteins

质子通道蛋白中的质子传导途径

• 批准号: 10244955
• 负责人: Huong Tran Kratochvil
• 金额: $ 9.72万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10244955
• 关键词: Acids; Address; Affect; Amantadine; Antiviral Agents; Binding; Bioenergetics; Biological Process; Biophysics; Cell membrane; Cells; Cellular Membrane; Charge; Crystallization; Crystallography; Diffusion; Disease; Drug resistance; Event; Glutamine; Hydration status; Hydrogen Bonding; Influenza; Influenza A virus; Ions; LGLA; Lead; Length; Lipid Bilayers; Measurement; Mediating; Membrane; Methods; Molecular; Molecular Conformation; Motion; Mutation; Pathway interactions; Permeability; Pharmaceutical Preparations; Phase; Play; Positioning Attribute; Process; Proteins; Protons; Replication-Associated Process; Resolution; Role; Signal Transduction; Site; Stream; Structure; Testing; Time; Vertebral column; Virus Replication; Water; Work; computer studies; deprotonation; design; infrared spectroscopy; molecular dynamics; mutant; novel; protonation; resistance mutation; transmission process; two-dimensional

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.

项目摘要： 质子通道蛋白增强了质子在细胞膜上的流动，并演变出细胞对照 超过质子的选择性和电导率，以有效地实现其功能，同时保持细胞完整性。 通过形成动态质子传导途径，这些传导途径模仿稀酸中观察到的水线 对于质子扩散，质子沿着氢键网络迅速和选择性地移动 散布在质子通道蛋白的腔内的狭窄水和可离子的侧链。一种方式质子 通道介导质子电导率是通过导向水线，该导管是稳定的腔内水。 通过极性蛋白质组。在高分辨率晶体中观察到的指导水线是经过充分研究的 结构，但是它们是移动还是静态以及它们的动态如何影响质子电导率 不清楚。调节质子选择性和电导率的另一种方法是通过瞬态水线 被认为是形成和消散的，以使质子通量通过包装饱满的Apolar片段。而瞬态水 电线已在分子动力学（MD）模拟中被假设，它们在根本上很难测试 实验。最后，质子通道还使用质子穿梭和去质子化的质子班车机制 通过可电离的侧链（例如他的GLU和ASP）调整质子电导，但目前尚不清楚 这些侧链介导孔溶剂化以及质子穿梭机构是否导致净运输 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）看似稳定的导水线和蛋白质极基团的氢键网络，（2）瞬态 水线和（3）由可离子的侧链组成的质子班车。 通过我们对天然质子通道的拟议研究，流感A基质蛋白2（M2）和从头开始 设计质子通道，我们将测试（1）指导和瞬时水线的假设 信道蛋白赋予其选择性并决定其传导质子的能力，（2）质子班车是 不仅要定义这些质子通道的传导速率，而且在 调节质子和水的渗透率。在AIM 1中，我们将检查指南水线是移动的还是 M2质子通道和疾病相关突变体上的多维红外光谱静态。我们的 在存在和不存在药物的情况下的测量将使我们能够确定这些动态如何 网络影响质子电导，以及它们如何通过药物结合和抗性突变而变化，即 确定新的抗病毒策略至关重要。在AIM 2中，我们测试了瞬态水线的假设 新型质子通道的新手设计和表征，具有不同长度的Apolar区域。在 R00阶段（AIM 3），我们研究了可离子的侧层如何增强孔隙水合并研究是否是否研究 质子化/去质子化事件导致质子和水的共转移。