Architecture of the Diffusion Barrier in the Nuclear Pore Complex

描述（由申请人提供）：核孔复合物（NPC）是真核生物中最重要的分子机器之一，因为它打开细胞细胞质和核质之间的多孔通道，并控制所有核-细胞质的交通和通讯。其最重要的结构特征是在其中心有一个鲜为人知的半透性扩散屏障，它对小至4纳米的细胞质蛋白质保持紧密密封，但打开后可方便运输各种形状和大小的颗粒，最大可达40纳米。这种柔性屏障是由一种名为FG核孔蛋白（FG nup）的丝状蛋白家族组成的，该家族在其天然功能状态下具有大的未折叠结构域，这些结构域被多个苯丙氨酸甘氨酸基序（FG结构域）装饰。我们提出的研究的具体目的是：1)表征两种不同类型的FG nup细丝的动态结构和分子内相互作用，它们锚定在NPC的三个不同位置；2)检验FG nup细丝结构域之间的分子间联系在NPC中心形成丝状网状结构的假设，这为蛋白质的被动扩散建立了尺寸选择屏障。该实验将结合生物化学、生物物理学、细胞生物学、结构（核磁共振）和分子建模技术来深入了解FG蛋白的动态行为、结构和功能。我们还将获得关于蛋白质无序结构域的动态行为和结构的基本知识。

DESCRIPTION (provided by applicant): The nuclear pore complex (NPC) is one of the most important molecular machines in eukaryotes because it gates the porous conduits between the cytoplasm and nucleoplasm of cells and controls all nucleo-cytoplasmic traffic and communication. Its most important architectural feature is a poorly understood semi-permeable diffusion barrier in its center that maintains a tight seal against cytoplasmic proteins as small as 4 nanometers in size, but opens to allow facilitated transport of particles of all shapes and sizes up to 40 nanometers in size. This flexible barrier is composed of a family of filamentous proteins named FG nucleoporins (FG nups) that feature large unfolded domains in their native functional state, which are decorated with multiple phenylalanine glycine motifs (FG domains). The specific aims of our proposed research are to 1) characterize the dynamic structure and intra-molecular interactions of FG domains representing two different types of FG nup filaments that are anchored at three different locales of the NPC, and 2) test the hypothesis that inter-molecular associations between FG domains of nups create a filamentous meshwork structure at the NPC center, which establishes the size-selective barrier to the passive diffusion of proteins. The proposed experiments will combine biochemical, biophysical, cell biological, structural (Nuclear Magnetic Resonance), and molecular modeling techniques to gain insight into the dynamic behavior, structure and function of the FG nups. We will also gain fundamental knowledge on the dynamic behavior and structure of disordered domains of proteins in general.