Molecular Mechanisms of Ion Transport

我的实验室对细胞维持离子稳态的基本分子机制感兴趣。 我们研究了两个特殊的系统，分别负责钾和锌的运输。总体目标是 这两个系统都是一样的，这是为了从结构上发展对运输的全面理解 以及热力学观点。我们将使用广泛的生物物理和生化 方法，包括冷冻-EM结构确定，体外生物物理测试功能 用于分析动力学的表征、单分子FRET和分子动力学模拟 分子。通过这种方式，我们的目标是为每个系统定义一个能源环境，并用 稳定中间体的实验结构以及对高能过渡态的评价 它们定义了运输路径。我们还试图了解底物专一性和 负责能量耦合和调节的变构耦合的结构元件 机械装置。正在调查的第一个系统是KdpFABC，它是一种有趣而不同寻常的混合系统， 与P型ATPase相关的ATP依赖泵和与K超家族相关的K通道 传送者。我们之前的工作已经定义了这种异构体的结构，并提出了一种高度 一种新的传输机制，其中K进入类通道亚基的选择性过滤器，运行40？ 通过膜嵌入的隧道，然后由能量依赖的泵状亚单位排出 举止。我们现在计划对定点突变体进行功能分析，以验证这一假设并采用新的 研究能量学的方法。第二个系统是来自阳离子扩散的锌氢逆向转运蛋白Y1iP 促进者超级大家庭。该家族的成员形成同源二聚体，具有多个离子结合位点，并 被认为是通过一种交替的访问机制发挥作用的。对于这个系统，我们描述了两个不同的 通过冷冻-EM分析和分子动力学模拟，确定了锌结合在分子链上的作用。 过渡。我们试图更好地理解单个锌结合位点的作用，被遮挡状态的性质， 内向态和外向态之间的转换以及质子在这一过程中的作用。我们还计划 研究这个家族的其他成员，以解决底物专一性和结构特征的基础 它们被认为是用来规范运输过程的。除了对这两个具体问题进行解释之外 运输机制，我们希望我们的工作将提供新的方式来思考运输超越 卡通和结构动画，将蛋白质动力学和能源景观地图结合起来，以 描述这些分子机器的行为。

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.