The dynamic mechanism of nuclear transport visualized at the atomic scale

家长奖摘要 大分子核质运输的唯一媒介是核孔复合体 (Npc)，由称为核孔素(Nups)的蛋白质组成。这些问题的具体机制 核孔素为核运输因子(NTR)连接的货物提供选择性扩散 假设涉及转运因子和苯丙氨基甘氨酸之间的相对较弱的相互作用 在某些核蛋白(FG-NUP)中发现的丰富(FG)重复区域提供选择性，而连续的 高度的FG重复区域动态无序为这一过程提供了足够的速度，有助于 选择性扩散。核运输和鼻咽癌的缺陷与许多 疾病，使它们成为一个重要的治疗目标，但由于我们的贫穷，仍未得到充分利用 对运输的机械论理解。在本文中，我们力求：(I)了解 对于不同的FG-NUP(具有不同的FG‘味道’)和不同的FG-NUP，这种机制的复杂性 核运输因素(NTR)；二)确定与潜在的 NTR的不同机制路线，以及NTR和非运输生物分子是如何 在NPC中受到歧视；以及III)如何理解纳米级集合结构的先驱 组件。我们使用蛋白质工程、核磁共振、SANS以及其他技术进行的综合研究 生物物理方法和分子动力学(MD)模拟提供经过验证的聚合工具 对于具有明确定义的(如果不寻常的)功能的IDP系统。因此，我们的目标是剖析 原子分辨率鼻咽癌中FG重复区域如何选择性地限制非特异性的扩散 大分子，同时允许NTR的有效交换，使用三种协同但非 重叠目标：(1)确定不同FG-Repeat口味和 NTR类型；(2)确定FG重复序列的集合结构及其如何改变 与非关税壁垒的相互作用；以及(3)确定不同的非关税壁垒在以下情况下如何移动 不同的FG重复口味。 这项研究的结果将是第一张原子尺度的功能角色的动态图片 通过描述FG重复的两种主要风格来重复区域，这些重复对大多数 全国人大的变化。这包括三个层次：i)不同FG重复中的主位和变异 具有不同NTR的类型；ii)不同FG重复类型的潜在聚合物性质；iii)如何 与其他类似材料相比，NTR的相对扩散得到了实际的增强。 这项研究的影响将是我们对 核运输机制。这些数据将为未来的翻译工作提供一条途径 在病变细胞中受到不利影响的地方，修改核运输。

PARENT AWARD ABSTRACT The sole mediators of nucleocytoplasmic transport of macromolecules are Nuclear Pore Complexes (NPCs), comprised of proteins termed nucleoporins (Nups). The specific mechanism by which these nucleoporins provide selective diffusion of nuclear transport factor (NTR)-linked cargoes is hypothesized to involve relatively weak interactions between transport factors and phenylalanyl glycyl rich (FG) repeat regions found in certain Nups (FG-Nups) to provide selectivity, while the continuous high degree of FG-repeat region dynamic disorder provides sufficient speed to the process, aiding selective diffusion. Defects in nuclear transport and the NPC are associated with numerous diseases, making them an important therapeutic target that is still underuseddue to our poor mechanistic understanding of transport. In this continuation, we seek to:(i) understand the complexities of this mechanism with various FG-Nups (with different ‘flavors’ of FGs) and different nuclear transport factors (NTRs); ii) establish the underlying properties associated with potential different mechanistic routes for NTRs, and how NTRs and non-transported biomolecules are discriminated in the NPC; and iii) pioneer how to understand the nanoscale ensemble structures of the components. Our integrative studies using protein engineering, NMR, SANS, as well as other biophysical methods and molecular dynamics (MD) simulation to provide paradigmatic tools validated for an IDP system with a well-defined (if unusual) function. Our goals are therefore to dissect at atomic resolution how FG repeat regions in the NPC selectively restrict diffusion of non-specific macromolecules while permitting the efficient exchange of NTRs, using three synergistic but non- overlapping Aims: (1) determine the specificities of interactions of different FG-repeat flavors and NTR types; (2) determine the ensemble structures of FG repeats and how they are altered on interaction with NTRs; and (3) determine how different NTRs move in the presence of ensembles of different FG repeat flavors. The outcome of this research will be the first atomic scale dynamic pictures of the functional roles of FG repeatregions by describing for the two major flavors of FG repeats contributing to most of the flux in the NPC. This isat three levels: i) themes and variations in the interaction of different FG repeat types with different NTRs; ii) underlying polymer properties of different FG repeat types; iii) how the actual enhancement of relative diffusionof NTRs compared to other similar materials comes about. The impact of this research will be a substantial increase in our knowledge of the detailed mechanisms of nuclear transport. These data will enable a route to future translational efforts to modify nuclear transport where it has been adversely affected in diseased cells.