The dynamic mechanism of nuclear transport visualized at the atomic scale

 描述(由申请人提供)：核孔复合体(NPC)形成一种选择性过滤器，允许运输因子(TF)及其货物快速通过。一类特殊的NUP(FG NUP)具有内在的无序区域，含有富含苯丙氨酰-甘氨酰的重复序列(FG NUP)，这些重复序列与TF相互作用，确保它们选择性地跨NPC易位。如何在分子水平上实现选择性转运仍不清楚，因为FG Nup行为和Tf-FG Nup相互作用的关键原子尺度细节一直缺失。我们将体外转运系统与核磁共振光谱相结合，以确定FG重复序列中所有残基的原子尺度行为。这些实验表明，在阻止其他大分子通过的同时。快速、选择性运输的潜在机制。我们证明了细胞环境在完全无序的状态下稳定了FG重复，形成了一种动态的“拴系流体”，并且TF上的多个苯丙氨酸结合位点与FG重复之间的许多频繁和瞬时的相互作用的综合作用提供了一个健壮的、可调节的和选择性的运输系统。综上所述，我们已经表明，我们可以确定选定的FG重复区域在其正常细胞环境和典型TF存在的情况下，结构和动力学的关键方面的动态原子尺度行为，明确地展示了这些方法的巨大和广泛的潜力。我们现在的目标是应用这些正交和高度创新的方法，主要是使用核磁共振，在三个水平上获得FG重复区域功能作用的完整原子尺度动态图像：(I)潜在的聚合物性质和环境影响；(Ii)与TF和其他大分子的相互作用，包括了解非特定大分子是如何被排除的；以及(Iii)运输NPC的特定拓扑和系综性质的影响。这项研究的影响将是大幅增加我们对通过鼻咽癌的运输的了解，探索具有病理意义的内毒素及其在细胞中的相互作用的新方法，以及将FG重复性质的原子尺度信息与运输现象直接联系起来。此外，很可能会产生新的方法来描述不可折叠的内在无序蛋白质的未知性质和功能，并对其他领域产生影响，例如淀粉样变性疾病。这一建议解决了生物学中选择性扩散领域的一个基本知识空白，这可能允许合理的治疗方法和与鼻咽癌相关的分子设计，例如肿瘤和病毒入侵。

 DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargo. A particular class of Nup (FG Nups) has intrinsically disordered regions containing phenylalanyl-glycyl rich repeats (FG Nups) that interact with TFs to ensure their selective translocation across the NPC. How selective transport is achieved at the molecular level remains unclear, because key atomic-scale details of FG Nup behavior and TF-FG Nup interactions have been missing. We have combined in vitro transport systems with NMR spectroscopy to determine the atomic-scale behavior of all residues in the FG repeats. These experiments reveal that while blocking the passage of other macromolecules. underlying mechanism of rapid, selective transport . We show that cellular milieux stabilize the FG repeats in a fully disordered state, forming a dynamic "tethered fluid", and that the combined effect of many frequent and transient interactions between multiple phenylalanine binding sites on the TFs and the FG repeats provides a robust, tunable and selective transport system. Taken together, we have shown that we can determine the dynamic atomic scale behavior of key aspects of the structure and dynamics of selected FG repeat regions in the presence of their normal cellular environment and typical TFs, definitively demonstrating the tremendous and broad-reaching potential of these approaches. Our objectives now are to apply these orthogonal and highly innovative approaches, primarily using NMR, to obtain complete atomic scale dynamic pictures of the functional roles of FG repeat regions at three levels: (i) underlying polymer property and environmental influences; (ii) interactions with TFs and other macromolecules, including understanding how non-specific macromolecules are excluded; and (iii) the influence of specific topology and ensemble properties of the transporting NPC. The impact of this research will be a substantial increase in our knowledge of transport through the NPC, novel methods for probing IDPs of pathological significance and their interactions in cells, and direct linkage of atomic scale information of FG repeat properties to transport phenomena. In addition, it is likely that new approaches to describing the unrecognized properties and functions of unfoldable intrinsically disordered proteins will be produced and impact in other areas, e.g. amyloidosis diseases. This proposal addresses a fundamental knowledge gap in the area of selective diffusion in biology, which may permit rational therapeutic approaches and molecular design related to the NPC for e.g. neoplasms, and viral invasion.