A considerable gap in our understanding of how cytoskeletal motor proteins function is the lack of a high resolution structure of the motor-filament interaction. The structural characterization of different components of the actomyosin system will provide insight into the mechanism of force production by myosin motors and will shed light on any conformational changes that occur within the actin filament upon motor binding. Due to the problems associated with the polymerization of wild-type actin at high concentrations necessary for crystallization, I will employ a novel molecular approach based on two complementary mutant actin monomers that can only oligomerize at one end. By combining these purified mutant actins, I will be able to isolate, crystallize, and solve the structure of the filamentous actin nucleation complex (trimer or tetramer of actin). I will improve the quality of existing crystals of two unconventional myosin motor domains from Dictyostelium discoideum, MyoD (class-l) and MyoJ (class-V), by adjusting purification and crystallization conditions and then solve the X-ray structures. Finally, I will characterize the actomyosin complex using X- ray crystallography, analytical ultracentrifugation and gel filtration, and isothermal titration calorimetry to obtain a high resolution structure of the motor-filament interaction. In muscle cells, the actomyosin system provides the force necessary for proper muscle contraction. Many diseases, such as heart disease and muscular distrophy, arise from improper functioning of the actomyosin complex. In non-muscle cells, the actomyosin system is central to many important biological functions, including cell motility and cell division. The persistence of cancer relies on the cell's ability to utilize the actomyosin machinery to power these vital biological processes. This study will lay the foundation for the structure-based design of chemotheraputic agents that disrupt the function of this necessary machine and prevent the metastasis of cancer cells. ? ? ?
| Cochran, Jared C; Zhao, Yu Cheng; Wilcox, Dean E et al. (2011) A metal switch for controlling the activity of molecular motor proteins. Nat Struct Mol Biol 19:122-7 |
| Zhao, Yu Cheng; Kull, F Jon; Cochran, Jared C (2010) Modulation of the kinesin ATPase cycle by neck linker docking and microtubule binding. J Biol Chem 285:25213-20 |
| Cochran, Jared C; Sindelar, Charles V; Mulko, Natasha K et al. (2009) ATPase cycle of the nonmotile kinesin NOD allows microtubule end tracking and drives chromosome movement. Cell 136:110-22 |
