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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 驱动蛋白-1是水解ATP以驱动细胞内货物运输的马达蛋白。它由两条重链（KHC）和两条轻链（KLC）组成。驱动蛋白-1的适当调节防止了马达的错误定位，并允许与其他分子马达协调。受调节的驱动蛋白-1对折，使得KLC和调节性KHC尾部结构域与酶活性KHC头部接触。这种KHC/KLC复合物对驱动蛋白-1的调节机制至关重要，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，我们将使用小角X射线散射（SAXS）以~ 3 nm的分辨率可视化溶液中受调节的驱动蛋白-1全酶的前所未有的结构。活性驱动蛋白-1具有显着的结构变异性，我们预计，只有一个旋转半径的整个扩展分子将获得。然而，调节构象似乎是一个紧凑和刚性的结构，因此，我们希望从SAXS获得有价值的信息，调节驱动蛋白-1的分子形状。结合化学交联研究，我们将使用SAXS结构来确定各种驱动蛋白-1元件在这个受调节的复合物中相互作用的位置。在我们的第一次会议期间，我们能够收集样品数据，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日使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.