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是一种马达蛋白，可以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可以防止马达的错误定位，并允许与其他分子马达协调。受调控的Kinesin-1被折叠成两半，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。为了克服这个问题，我们将使用小角X射线散射(SAXS)来显示在~3 nm分辨率的溶液中调节的Kinesin-1全酶的前所未见的结构。活性Kinesin-1具有显著的结构变异性，我们预计只会获得整个扩展分子的旋转半径。然而，受调控的构象似乎是一个紧凑和刚性的结构，因此，我们希望从SAXS中获得关于受调控的kinesin-1的分子形状的有价值的信息。结合化学交联研究，我们将使用SAXS结构来确定各种Kinesin-1元件在这个受调控的复合体中相互作用的位置。在我们的第一次会议上，我们能够收集样本数据，但随后的分析确定，相机长度太短，无法确定最佳结构。我们计划在11月21日用更长的相机重复我们的实验，我们预计这将产生所需的数据。

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 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 mislocalization of the motor and allows for coordination with other molecular motors. Regulated kinesin-1 is folded in half 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 will use small-angle X-ray scattering (SAXS) to visualize the never-before-seen structure of a regulated kinesin-1 holoenzyme in solution at ~3nm resolution. Active kinesin-1 has significant structural variability, and we expect that only a radius of gyration for the entire extended molecule will be obtained. However, the regulated conformation appears to be a compact and rigid structure, therefore, we expect to obtain valuable information about the molecular shape of regulated kinesin-1 from SAXS. Together with chemical crosslinking studies, we will use the SAXS structure to determine where the various kinesin-1 elements interact with each other in this regulated complex. During our first session, we were able to collect sample data but subsequent analysis determined that the camera length was too short for optimum structure determination. We are scheduled to repeat our experiments with a longer camera on Nov 21, and we anticipate that this will yield the desired data.