DESCRIPTION (provided by applicant): Motor proteins play a critical role in intracellular transport and motility, which are required for several basic cellular processes such as mitosis. Dyneins are large, complicated, microtubule-based, minus-end directed motor proteins belonging to the AAA (ATPases Associated with diverse cellular Activities) family of enzymes. Due to the critical roles cytoplasmic and axonemal dyneins play in eukaryotic cells, defects in their function have been linked to a variety of pathologies including neurodegenerative diseases and cancer. The details of how dynein dysfunction leads to disease states remain obscure, in large part due to our limited understanding of the molecular mechanism by which dynein functions as a motor protein. Recent and ongoing advances in structural biology and microscopy techniques make it an exciting and ideal time to probe the structural and mechanistic basis of dynein motility in greater detail. My current expertise as a structural biologist is in the areas of X-ray crystallography and NMR, two excellent tools to study how structure an dynamics come together to facilitate function in biological macromolecules. During the initial phase of my postdoc, I have had some training in electron microscopy, which I have used to study the allosteric mechanism of yeast cytoplasmic dynein motility. I would now like to extend this training to become an expert in the rapidly advancing field of electron cryomicroscopy (cryo-EM). Recently, cryo-EM structures have been reported at extremely high resolutions, making it a phenomenal tool with which to study how large protein complexes work, which is one of my long-term interests. Additionally, I would like to gain complementary expertise in using single-molecule light microscopy to study protein dynamics in solution. This training will provide me with a unique tool kit that equips me to study structure-dynamics-function relationships of biological systems from many different perspectives. The broad goal of the proposal is to dissect the structure, dynamics and function of cytoplasmic and axonemal dyneins. Accordingly, the specific aims are to: 1) Probe the dynamics and functional role of dynein's stalk domain 2) Determine high-resolution structure of the full-length axonemal dynein complex 3) Recombinantly generate axonemal dynein to study mutants at the single molecule level 4) High-resolution structural analyses of dynein-microtubule complexes This work will provide fundamental insights into the structure-dynamics-function relationship in dynein, thus setting the stage for further molecular studies of disease-related mutants and the role of dynein in cellular function and disease.

PUBLIC HEALTH RELEVANCE: Dynein is a large motor protein that moves along cytoskeletal microtubules and facilitates cellular processes such as ciliary beating as well as transport of intracellular cargos such as proteins, RNA and organelles. When dysfunctional, several dynein-mediated processes are associated with brain abnormalities, heart defects, neurodegenerative disorders and cancer. The results of proposed study will yield insights into the mechanism of dynein-based motility, and possibly how the motor protein complex might be exploited for the development of small molecule drugs.