The major goal of this grant is to understand the role of actin in producing cytoplasmic movement and to provide structural insights on the protein:protein interactions that link signal transduction pathways to the actin-based cytoskeleton. A 1.7 Angstrom resolution structure of the profilin:actin complex will allow the elucidation of water-mediated networks in the interdomain cleft of the actin molecule. A crystal structure of human profilin II will help explain why the brain form of this abundant actin-binding protein binds poly-L-proline more tightly than human profilin I. Structures of plant and mammalian profilins with bound proline-rich sequences will provide important structural parameters on links between signalling proteins and the actin cytoskeleton. The purification of short oligomers of filamentous actin suitable for x-ray crystallography will set the stage for a structure determination of the most physiologically relevant form of actin. Actin is found in high abundance in all eukaryotic cells. More than 100 different proteins bind actin to regulate polymerization or to control its role or position in various cytoskeletal structures. High resolution structural data on the profilin:poly-L-proline interaction will be important for drug design efforts in the cases of diseases where cell motility or cortical shape changes play a role (cancer, hypercoagulable platelets, lymphocytosis, viral infections, growth cone neuropathies) and for understanding the formation of organs and limbs. Many bacterial and viral pathogens exploit the actin-rich cytoskeleton to invade eukaryotic cells, and knowledge of these mechanisms will enable new therapeutic models to be developed. Actin is a major constituent of muscle cells of all forms (cardiac, skeletal, smooth), and it is essential to have the highest resolution structures possible in order to understand in chemical terms how muscle performs work at high efficiency through a broad range of load and speed. Neurons and glial cells have high concentrations of actin at their dynamic peripheries and brain profilin II serves as a link between surface activity and the cytoskeleton. Knowledge of these structures will profoundly impact neurobiology. Profilin from plant pollen is a potent allergen. Structures of plant profilin complexed with endogenous flavonols could lead to improved molecular level understanding of the IgE response in allergic individuals.
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