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： 质子通道蛋白增强质子穿过细胞膜的流动，并已进化出精细控制 超过质子选择性和电导率，以有效实现其功能，同时保持细胞完整性。 通过形成模拟稀酸中观察到的水线的动态质子传导路径 对于质子扩散，质子沿着由以下物质组成的氢键网络快速且选择性地移动 受限水和可电离侧链分散在质子通道蛋白的腔内。单向质子 通道通过引导水线介导质子传导性，引导水线是组织稳定的管腔水 由极性蛋白质基团组成。导水线经过充分研究，因为它们是在高分辨率晶体中观察到的 结构，但它们是移动的还是静态的以及它们的动力学如何影响质子传导性仍然存在 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，它们是 被认为形成和消散以允许质子通量通过填充良好的非极性部分。虽有短暂的水 电线已在分子动力学（MD）模拟中假设，但它们基本上很难测试 实验性地。最后，质子通道还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，例如 His、Glu 和 Asp，来调节质子电导，但目前尚不清楚 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传输 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) 导水线和蛋白质极性基团看似稳定的氢键网络，(2) 瞬态 水线，以及（3）由可电离侧链组成的质子穿梭机。 通过我们对天然质子通道、甲型流感基质蛋白 2 (M2) 和 de novo 的研究 设计质子通道后，我们将测试以下假设：(1) 质子内的引导和瞬态水线 通道蛋白赋予其选择性并决定其传导质子的能力，并且（2）质子穿梭机是 不仅对于定义这些质子通道的传导率是必要的，而且在 调节质子和水的渗透性。在目标 1 中，我们将检查导水线是否是可移动的或 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检测。我们的 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态如何 网络影响质子电导，以及它们如何随着药物结合和耐药突变而变化，这是 对于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 R00 阶段（目标 3），我们研究了可电离侧链如何增强孔隙水合作用，并研究是否 质子化/去质子化事件导致质子和水的共易位。