Allosteric Role of Dynein Light Chains in Dynein Assembly and Regulation

细胞质动力蛋白是一种基于微管的分子马达，其使用来自ATP水解的能量将细胞货物向微管的负端移动和运输。这种转运在细胞行为的几个方面是必不可少的，包括细胞迁移和分裂，染色体分离和囊泡运输。动力蛋白重链亚基与微管结合并含有提供动力的ATP位点，而轻链和中间链被认为与货物结合并调节马达的活性。来自Barbar实验室的最新数据表明，轻链LC 8的作用是促进动力蛋白中间链的天然无序N-末端结构域的二聚化，而不是如通常认为的那样主要用作货物衔接子。轻链Tctex-1是LC 8的结构同源物，并在与LC 8结合位点相邻的位点结合中间链，可能具有与LC 8相似的作用。该研究项目将测试轻链LC 8和Tctex-1的串联作用是促进动力蛋白组装和与货物适配器dynactin的相互作用，而不是作为货物适配器本身的假设。使用生物物理方法的组合，包括核磁共振光谱（NMR），等温滴定量热法和X射线晶体学，Barbar实验室将阐明两条轻链与动力蛋白中间链相互作用的三个重要结果的机制：1）它们如何有助于形成紧密的动力蛋白组装体，2）它们如何促进中间链的二聚化，以及3）它们如何可能促进中间链与动力肌动蛋白的p150 Glued亚基的更紧密的相互作用。这些轻链的重要作用将在该项目的合作者Tom Hays教授的实验室中进行体内测试。这些研究将为动力蛋白运动功能的结构生物学和热力学提供新的见解，并将补充在细胞内运输领域占主导地位的细胞生物学方法。由于这些研究提供了第一个结构的洞察力的无序到有序的转变中的动力蛋白组装和它的关联与dynactin的作用，他们允许有机会开发一个易于处理的系统来探测的作用，内在的障碍组装的大分子复合物。 更广泛的影响：PI在本科生的研究贡献以及指导少数民族和残疾学生方面有着良好的记录。PI正在她的校园中发挥主导作用，通过提供仪器和培训研讨会向研究界介绍分子生物物理学。此外，PI将发挥积极作用，通过提供研究实验室经验的机会，并通过开发教学材料和周六学院计划中的实践模块，促进高中生的基础科学。后者是俄勒冈州州立大学的一个非常成功的项目，旨在为高中生提供生物化学和生物物理学方面的服务。

Cytoplasmic dynein is a microtubule-based molecular motor that uses the energy derived from ATP hydrolysis to move and transport cellular cargo towards the minus ends of microtubules. This transport is essential in several aspects of cell behavior including cell migration and division, chromosome segregation, and vesicle trafficking. The dynein heavy chain subunits bind to microtubules and contain the ATP sites that provide the motive force, while the light and intermediate chains are thought to bind to cargo and regulate the activity of the motor. Recent data from the Barbar lab have suggested that the role of light chain LC8 is to facilitate dimerization of the natively disordered N-terminal domain of dynein intermediate chain, rather than to primarily function as a cargo adaptor, as commonly thought. Light chain Tctex-1, which is a structural homolog of LC8 and binds the intermediate chain at a site contiguous to the LC8 binding site, is likely to have a similar role as LC8. The research project will test the hypothesis that the tandem roles of light chains LC8 and Tctex-1 are to promote dynein assembly and the interaction with the cargo adaptor dynactin, and not to act as cargo adaptors themselves. Using a combination of biophysical approaches including nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry and X-ray crystallography, the Barbar lab will elucidate the mechanisms of three important outcomes of the interactions of both light chains to dynein intermediate chain: 1) how they contribute to formation of a tight dynein assembly, 2) how they promote dimerization of the intermediate chain, and 3) how they may promote a tighter interaction of the intermediate chain with p150Glued subunit of dynactin. The essential roles of these light chains will be tested in vivo in the lab of Professor Tom Hays, a collaborator on this project. The studies should provide novel insights into the structural biology and thermodynamics of dynein motor function, and will complement the cell biological approaches that predominate in the field of intracellular transport. Since these studies provide the first structural insights into the role of the disorder-to-order transition in dynein assembly and its association with dynactin, they allow the opportunity to develop a tractable system to probe the role of intrinsic disorder in the assembly of large macromolecular complexes. Broader Impacts: The PI has a strong record of research contributions with undergraduate students, and of mentoring minorities and students with disabilities. The PI is taking a leading role on her campus to introduce molecular biophysics to the research community by providing access to instrumentation and training workshops. Furthermore, the PI will take an active role to promote basic science among high school students by providing opportunities for research lab experience, and by developing instructional material and a hands-on module in the Saturday Academy program. The latter is a highly successful program at Oregon State University for outreach for high school students in which biochemistry and biophysics have not yet been represented.