RUI: Exploring the Structural Basis of Dynein Regulation

RUI：探索动力蛋白调节的结构基础

• 批准号: 2003557
• 负责人: Nikolaus Loening
• 金额: $ 29.7万
• 依托单位: Lewis and Clark College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2003557&HistoricalAwards=false
• 关键词: RUI; Exploring; Structural; Basis; Dynein

Motor proteins serve a number of functions in the cell, including helping transport biological molecules (cargo) to where they belong. One such motor protein, dynein, is important for moving cargo from the periphery of cells toward the center. In human cells, dynein needs to partner with another protein (dynactin) in order to move cargo across long distances, such as along nerve cells. The control or regulation of how these two proteins interact determines what, when, and where cargo are transported in a cell. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Nikolaus Loening from Lewis & Clark College to determine how the interactions between these two proteins are regulated by changes in the structure of dynein. Dr. Loening focuses on dynein intermediate chain, a part of the motor protein that initiates binding with dynactin, and studies how variations in its sequence and the addition of phosphate chemical groups change its binding behavior. The intermediate chain is difficult to study because it does not normally adopt a fixed and rigid shape; it is intrinsically disordered. Consequently, a variety of biophysical techniques are used to study the effects of these structural variations on the intermediate chain’s shape and its interactions with dynactin. This project provides undergraduate and high school students with opportunities to participate directly in this research. In addition, some parts of this research are incorporated into laboratory and special topics classes at Lewis & Clark College, thereby broadening participation in this project to include a larger number of undergraduate students. This research project characterizes the structural properties of a little-studied isoform of the dynein intermediate chain (IC) to better localize the interaction between IC and its binding partners. The objective of this project is to develop a structural model for how an intrinsically disordered region of IC binds with the p150Glued subunit of dynactin. Results from these studies lead to insights into how this interaction is regulated by the isoform type or phosphorylation state of dynein. The dynamic nature of this system makes it difficult to study by cryo-electron microscopy or X-ray crystallography. In addition, the complex has characteristics that make it unfavorable for study by conventional NMR methods. This project uses a combination of mutagenesis to stabilize p150Glued combined with better localization of the binding site to allow the development of smaller, more tractable constructs that are providing high-resolution structural information. This work helps to develop an understanding for how this system is regulated, which further complements studies on the less dynamic regions of the dynein complex. In the course of this research, new methods for protein NMR spectroscopy are developed to benefit not only researchers studying dynein and intrinsically disordered proteins, but the protein NMR community at large.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

马达蛋白在细胞中起着许多作用，包括帮助运输生物分子（货物）到它们所属的地方。动力蛋白就是这样一种运动蛋白，它对于将货物从细胞外围向中心移动非常重要。在人类细胞中，动力蛋白需要与另一种蛋白质（动力肌动蛋白）合作，以便长距离移动货物，例如沿着神经细胞。这两种蛋白质如何相互作用的控制或调节决定了细胞中运输什么、何时和何处的货物。有了这个奖项，化学部的生命过程化学计划正在资助刘易斯克拉克学院的Nikolaus Loening博士，以确定这两种蛋白质之间的相互作用是如何通过动力蛋白结构的变化来调节的。Loening博士专注于动力蛋白中间链，这是启动与dynactin结合的马达蛋白的一部分，并研究其序列的变化和磷酸盐化学基团的添加如何改变其结合行为。中间链很难研究，因为它通常不采用固定和刚性的形状;它本质上是无序的。因此，各种生物物理技术被用来研究这些结构变化对中间链的形状及其与动力蛋白的相互作用的影响。该项目为本科生和高中生提供了直接参与这项研究的机会。此外，本研究的某些部分被纳入刘易斯克拉克学院的实验室和专题课程，从而扩大了对本项目的参与，以包括更多的本科生。该研究项目表征了动力蛋白中间链（IC）的一个很少研究的异构体的结构特性，以更好地定位IC与其结合伙伴之间的相互作用。本项目的目的是建立一个结构模型，用于研究IC的内在无序区域如何与动力蛋白的p150 Glued亚基结合。从这些研究的结果导致深入了解这种相互作用是如何调节的动力蛋白的亚型或磷酸化状态。该系统的动态性质使得难以通过低温电子显微镜或X射线晶体学进行研究。此外，该配合物具有使其不利于通过常规NMR方法进行研究的特性。该项目使用诱变的组合来稳定p150 Glued，结合结合位点的更好定位，以允许开发更小，更易于处理的结构，提供高分辨率的结构信息。这项工作有助于了解该系统是如何调节的，这进一步补充了对动力蛋白复合物动态较小区域的研究。在这项研究的过程中，开发了新的蛋白质核磁共振光谱方法，不仅使研究动力蛋白和内在无序蛋白的研究人员受益，而且使整个蛋白质核磁共振社区受益。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。