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

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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.