We will determine how chemical energy is transformed into mechanical energy by two types of molecular motors, those of the myosin and kinesin superfamilies. The goal of understanding the mechanism of biological motors requires: a) a determination of their atomic resolution structures, b) knowledge of how these structures change in specific states and c) knowledge of how these structures fit into the cycle. Our approach will build on our previous work which determined the structures of two members of the kinesin motors, kinesin and NCD, and the NCD dimer. These structures will now enable us to design rational experiments, mutations, placement of probes, etc., to determine how they function. The structures of kinesin/ncd complexed with nucleotides/nucleotide analogs remain a critical missing piece, and we will obtain these structures. In addition we will determine the structure of the motor region of another motor family, dynein. The structures of the motor proteins complexed with their polymers are crucial to understanding their function. These structures will be defined by lower resolution techniques, spectroscopy, electron microscopy, etc. We will also attempt to form crystals between the motors and oligomers of the polymers. Conformational changes in the proteins, and the energetic differences between conformations will be monitored using spectroscopic probes, both fluorescent and paramagnetic. We will concentrate on specific elements that transmit structural changes in the catalytic domains to the neck, or stalk, which in turn amplifies these conformational changes to produce force and motion. In myosin these elements include a deep cleft, the converter region that transmits conformational changes to the neck, and the neck region, which may act as a lever system. In kinesin these elements include the switch II region, and the interface between the motor domain and the neck, as well as the stalk adjacent to the neck. The proposed work involves a combination of molecular biology protein biochemistry, x-ray crystallography, and biophysical techniques. We have brought together a unique group of investigators that can extend our knowledge in each of these areas leading to a better picture of the molecular mechanism of these two motors that produce force and motion in all eukaryotic cells.
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