Molecular Mechanisms of Ion Transport - Equipment supplement

离子传输的分子机制 - 设备补充

• 批准号: 10798994
• 负责人: David L. Stokes
• 金额: $ 8.98万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798994
• 关键词: ATP phosphohydrolase; Address; Adopted; Architecture; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biophysics; Cartoons; Cations; Cells; Coupling; Cryoelectron Microscopy; Diffusion; Elements; Equipment; Family member; Goals; Homeostasis; Hybrids; In Vitro; Individual; Investigation; Ion Transport; Ions; Laboratories; Light; Maps; Measures; Membrane; Molecular; Molecular Conformation; Molecular Machines; Nature; Pathway interactions; Potassium Channel; Process; Protein Dynamics; Proteins; Protons; Pump; Role; Site; Structure; Substrate Specificity; System; Thermodynamics; Transport Process; Travel; Work; Zinc; animation; antiporter; instrument; interest; molecular dynamics; mutant; novel; novel strategies; simulation; single-molecule FRET

My laboratory is interested in fundamental molecular mechanisms by which cells maintain ionic homeostasis. We study two particular systems responsible for transport of K+ and Zn2+, respectively. Overall goals are the same for both systems, which are to develop a comprehensive understanding of transport from structural as well as thermodynamic perspectives. We will use a broad spectrum of biophysical and biochemical approaches, including cryo-EM for structure determination, in vitro biophysical assays for functional characterization, single-molecule FRET and Molecular Dynamic simulations for analyzing dynamics of the molecules. In this way, we aim to define an energy landscape for each system, annotated with the experimental structures for stable intermediates as well as an appreciation for the high-energy transition states that define the transport pathway. We also seek to understand determinants of substrate specificity and structural elements responsible for the allosteric coupling that underlies energy coupling and regulatory mechanisms. The first system under investigation is KdpFABC, an interesting and unusual hybrid between an ATP-dependent pump related to P-type ATPases and a K+ channel related to the Superfamily of K+ Transporters. Our previous work has defined the architecture of this heterotetramer and suggests a highly novel mechanism for transport, in which K+ enters the selectivity filter of the channel-like subunit, travels 40 Å through a membrane-embedded tunnel, and is then expelled by the pump-like subunit in an energy-dependent manner. We now plan functional analyses of site-directed mutants to validate this hypothesis and to adopt new approaches to study the energetics. The second system is YiiP, a Zn2+/H+ antiporter from the Cation Diffusion Facilitator superfamily. Members of this family form homodimers, have multiple ion binding sites and are thought to function via an alternating access mechanism. For this system, we have characterized two different conformations by cryo-EM analysis and MD simulation, and have identified a role for Zn2+ binding on the transition. We seek to understand better the role of individual Zn2+ binding sites, the nature of occluded states, the transition between inward- and outward-facing states and the role of protons in this process. We plan also to study additional members of this family to address the basis for substrate specificity and structural features that have been postulated to regulate the transport process. In addition to shedding light on these two specific transport mechanisms, we hope that our work will offer new ways to think about transport that goes beyond cartoons and structural animations to incorporate protein dynamics and mapping of the energy landscape to describe the behavior of these molecular machines.

我的实验室对细胞维持离子稳态的基本分子机制感兴趣。 我们分别研究了负责K+和Zn2+运输的两个特定系统。总体目标是 这两个系统都相同，这些系统都可以对从结构性的运输进行全面了解 以及热力学观点。我们将使用广泛的生物物理和生化范围 方法，包括用于结构确定的冷冻EM，用于功能的体外生物物理测定 表征，单分子fret和分子动态模拟，用于分析动力学 分子。通过这种方式，我们旨在为每个系统定义一个能源景观，并用 稳定中间体的实验结构以及对高能过渡状态的欣赏 这定义了运输路径。我们还试图了解确定底物特异性和 负责能量耦合和调节的变构耦合的结构元素 机制。正在研究的第一个系统是KDPFABC，这是一种有趣且不寻常的混合体 与P型ATPases有关的ATP依赖性泵和与K+超家族有关的K+通道 运输商。我们以前的工作已经定义了该异光聚体的架构，并提出了高度的 新型运输机制，其中K+进入了类似通道的亚基的选择性滤波器40Å 通过膜包裹的隧道，然后在能量依赖性的泵状亚基探索 方式。现在，我们计划对现场定向突变体的功能分析来验证这一假设并采用新的假设 研究能量学的方法。第二个系统是Yiip，是阳离子扩散的Zn2+/H+抗逆物 促进者超家族。该家族成员形成同型二聚体，具有多个离子绑定位点，并且 被认为是通过替代访问机制运行的。对于这个系统，我们表征了两个不同的 通过冷冻EM分析和MD模拟构象，并确定了Zn2+结合的作用 过渡。我们试图更好地理解单个Zn2+结合位点的作用，封闭状态的本质， 向内和向外的状态与质子在此过程中的作用之间的过渡。我们也计划 研究该家族的其他成员以解决底物特异性和结构特征的基础 已经假定的以调节运输过程。除了阐明这两个特定 运输机制，我们希望我们的工作将提供新的方式来思考超越的运输 漫画和结构动画，以结合蛋白质动力学和能量景观的映射 描述这些分子机器的行为。