SAXS STUDY OF REGULATION OF THE KINESIN-1 MOTOR BY THE KINESIN LIGHT CHAINS

驱动蛋白轻链对驱动蛋白-1 马达调节的 SAXS 研究

• 批准号: 8168626
• 负责人: Sarah E. Rice
• 金额: $ 0.54万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168626
• 关键词: ATP Hydrolysis; Annual Reports; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Elements; Funding; Grant; Head; Holoenzymes; Institution; Intracellular Transport; Kinesin; Length; Light; Modeling; Molecular Conformation; Molecular Motors; Motor; Nature; Process; Proteins; Regulation; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Sampling; Solutions; Source; Structure; Tail; Techniques; United States National Institutes of Health; insight; molecular shape; prevent; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kinesin-1 is a ubiquitous motor protein that hydrolyzes ATP to drive the transport of intracellular cargo. It is composed of two heavy chains (KHCs) and two light chains (KLCs). Proper regulation of kinesin-1 prevents its mislocalization and allows for coordination with other molecular motors. Regulated kinesin-1 is folded over on itself so that the KLCs and regulatory KHC tail domains come in contact with the enzymatically active KHC heads. This KHC/KLC complex is central to the regulatory mechanism of kinesin-1, yet the structure remains undetermined because the complex is not amenable to traditional structure determination techniques. To overcome this, we used small-angle X-ray scattering (SAXS) to visualize the never-before-seen structure of a regulated kinesin-1 holoenzyme in solution at ~3nm resolution. The regulated conformation of kinesin-1 appears to be a compact and rigid structure, therefore we expect to obtain valuable information about its molecular shape from SAXS. We will use the SAXS structure to determine where the various kinesin-1 elements interact with each other in this regulated complex. Since our previous annual report, we have successfully collected the sample data and concluded our experiments. The raw data is currently being processed and analyzed, and we expect the final model to provide significant insight into the nature of kinesin-1 regulation. We were also able to extend our original studies by performing SAXS on complexes of head and tail fragments. The smaller size of these fragments allowed us to achieve higher SAXS resolution compared to the full-length molecule.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Kinesin-1 是一种普遍存在的运动蛋白，可水解 ATP 以驱动细胞内货物的运输。它由两条重链 (KHC) 和两条轻链 (KLC) 组成。正确调节 kinesin-1 可以防止其错误定位并允许与其他分子马达协调。受调节的驱动蛋白-1 自身折叠，以便 KLC 和调节 KHC 尾部结构域与具有酶活性的 KHC 头部接触。这种 KHC/KLC 复合物是驱动蛋白-1 调节机制的核心，但其结构仍未确定，因为该复合物不适合传统的结构测定技术。为了克服这个问题，我们使用小角 X 射线散射 (SAXS) 以约 3 nm 的分辨率可视化溶液中受调节的驱动蛋白-1 全酶的前所未见的结构。驱动蛋白-1的受调控构象似乎是紧凑且刚性的结构，因此我们期望从SAXS获得有关其分子形状的有价值的信息。我们将使用 SAXS 结构来确定各种 kinesin-1 元件在该调节复合物中相互作用的位置。自上一份年度报告以来，我们已经成功收集了样本数据并完成了我们的实验。目前正在处理和分析原始数据，我们期望最终模型能够对驱动蛋白-1 调控的本质提供重要的见解。我们还能够通过对头部和尾部片段的复合物进行 SAXS 来扩展我们最初的研究。与全长分子相比，这些片段较小的尺寸使我们能够实现更高的 SAXS 分辨率。