The dynamic mechanism of nuclear transport visualized at the atomic scale

 描述（由申请人提供）：核孔复合物（NPC）形成选择性过滤器，允许转运因子（TF）及其货物快速通过。一类特殊的Nup（FG Nups）具有包含富含苯丙氨酰-甘氨酰重复序列（FG Nups）的内在无序区域，这些重复序列与TF相互作用，以确保它们选择性地穿过NPC移位。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。我们结合了体外转运系统与NMR光谱，以确定原子尺度的行为中的FG重复的所有残基。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。我们表明，细胞环境稳定的FG重复在一个完全无序的状态，形成一个动态的“拴系流体”，和许多频繁和短暂的相互作用之间的相互作用的TF和FG重复的多个苯丙氨酸结合位点提供了一个强大的，可调的和选择性的运输系统的综合效果。总之，我们已经表明，我们可以确定在其正常细胞环境和典型TF的存在下，所选FG重复区域的结构和动力学的关键方面的动态原子尺度行为，明确地证明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NMR，以获得完整的原子尺度动态图片的功能作用的FG重复区在三个层次：（i）潜在的聚合物性质和环境的影响;（ii）相互作用与TF和其他大分子，包括了解如何排除非特异性大分子;以及（iii）传输NPC的特定拓扑结构和系综性质的影响。这项研究的影响将是一个实质性的增加，我们的知识运输通过NPC，新的方法探测IDP的病理意义和它们在细胞中的相互作用，并直接链接的原子尺度信息FG重复属性的运输现象。此外，很可能会产生新的方法来描述未被识别的属性和功能的非折叠的内在无序蛋白质，并在其他领域的影响，如淀粉样变性疾病。该提案解决了生物学中选择性扩散领域的基本知识空白，这可能允许与NPC相关的合理治疗方法和分子设计，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.