Bridge 5: Conformational Dynamics in the CLC Channel/Transporter Family

作为次级活性转运蛋白，CLC利用储存在一个离子梯度（Cl-）中的能量。 或H+）以逆着其电化学梯度泵送另一个离子。这种情况发生在紧 蛋白质构象变化与离子结合、解结合和易位事件的偶联。 几种CLC同系物的X射线晶体学结构提供了一个宝贵的结构 为理解这个家族的分子机制奠定了基础。尽管作出了广泛的努力， 然而，结晶仅揭示了一种基本CLC构象状态。在这个项目中， 我们将描述未知的构象状态，并描述内部的动力学 这些构象状态以及状态之间的结构转变， 共同产生CLC转运蛋白功能。在目标1中，我们将表征分子 详细的难以捉摸的面向外（OF）和面向内（IF）的国家，通过使用双 电子-电子共振（DEER）测量位置对之间的距离变化， CLC-ec 1上的定向自旋标记，这是一种研究得很好的同系物，其封闭的结构 国家已经确定。计算建模将指导DEER实验设计， 实验结果将指导结构模型的细化和验证建模 预测。结晶的国家将接近使用状态稳定的交联 基于结构模型设计，以及构象特异性合成抗原 可用作结晶伴侣的粘合剂。在目标2中，我们将研究三个 状态内和状态之间的构象动力学方面，这是阐述 整体CLC运输机制。首先，我们将描述水动力学和H+- 运输途径在不同的构象状态之间变化，使用扩展的分子 动力学（MD）模拟与实验验证相结合。第二，我们将确定 运输循环中的潜在中间体，并评估状态之间转换的动力学 使用单分子荧光共振能量转移（smFRET）测量， 急冻急冷鹿。最后，详细研究离子的结合和移位 与蛋白质构象变化相结合，我们将模拟状态之间的转换。 先进的非平衡模拟和采样技术将被用来描述 主要状态（IF，OF和闭塞）之间的过渡路径，并计算自由 与这些转变相关的能量分布。

As secondary active transporters, CLCs harness energy stored in one ion gradient (Cl- or H+) to pump the other ion against its electrochemical gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. The X-ray crystallographic structures of several CLC homologs provide an invaluable structural foundation for understanding molecular mechanisms in this family. Despite extensive efforts, however, crystallization has revealed only one basic CLC conformational state. In this project, we will characterize the unknown conformational states and describe both the dynamics within these conformational states as well as the structural transitions between the states, which together give rise to CLC transporter function. In Aim 1, we will characterize the molecular details of the elusive outward-facing (OF) and inward-facing (IF) states by using double electron-electron resonance (DEER) to measure distance changes between pairs of site- directed spin labels on CLC-ec1, a well-studied homolog for which the structure of the occluded state has been determined. Computational modeling will guide DEER experimental design, and experimental results will guide the refinement of the structural models and validate modeling predictions. Crystallization of states will be approached using state-stabilizing cross-links designed based on the structural models, as well as conformation-specific synthetic antigen binders that can be used as crystallization chaperones. In Aim 2, we will investigate three aspects of conformational dynamics within and between states that are key to elaborating the overall CLC transport mechanism. First, we will characterize how water dynamics and H+- transport pathways vary amongst the different conformational states using extended molecular dynamics (MD) simulations combined with experimental validation. Second, we will identify potential intermediates in the transport cycle and evaluate kinetics of transitions between states using single-molecule fluorescence resonance energy transfer (smFRET) measurements and rapid-freeze quench DEER. Finally, to investigate in detail how binding and translocation of ions are coupled to protein conformational changes, we will model transitions between the states. Advanced non-equilibrium simulations and sampling techniques will be used to describe the transition pathways between the major states (IF, OF, and occluded) and to calculate the free energy profiles associated with these transitions.