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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Kinesin-1是一种普遍存在的运动蛋白，它能水解三磷酸腺苷来驱动细胞内货物的运输。它由两条重链(KHC)和两条轻链(KLCS)组成。适当地调节Kinesin-1可以防止其错误定位，并允许与其他分子马达协调。受调控的Kinesin-1自身折叠，从而使KLCS和受调控的KHC尾部结构域与具有酶活性的KHC头部接触。这种KHC/KLC复合体是Kinesin-1调控机制的核心，但由于该复合体不适用于传统的结构测定技术，其结构仍未确定。为了克服这一问题，我们使用小角X射线散射(SAXS)在~3 nm分辨率下显示了调节的Kinesin-1全酶在溶液中从未见过的结构。Kinesin-1的调控构象看起来是一个紧凑和刚性的结构，因此我们希望从SAXS中获得关于其分子形状的有价值的信息。我们将使用SAXS结构来确定在这个受调控的复合体中各种kinesin-1元件相互作用的位置。自上一次年报以来，我们已经成功地收集了样本数据并结束了我们的实验。原始数据目前正在处理和分析中，我们预计最终的模型将为Kinesin-1调控的性质提供重要的见解。我们还能够通过对头部和尾部碎片的复合体进行SAXS来扩展我们的原始研究。这些片段的较小尺寸使我们能够获得比全长分子更高的SAXS分辨率。

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.