STRUCTURAL STUDIES OF MOTOR PROTEINS

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Description (taken from the applications): Our project is to determine atomic level structures for kinesin family motor proteins. We will use single crystal X-ray crystallography to determine structures of protein complexes of kinesins with proteins, peptides or small molecules that induce the kinesin molecule to change its structure from the ADP-kinesin and ADP-NCD complexes already imaged by X-ray diffraction. Our experiments will focus on three kinesins: two are molecular motors and one is a microtuble disassembly machine. NCD, the reverse traveling motor required to carry out meiosis and human kinesin, the forward traveling motor that delivers vesicles in neurons are to be crystallized with ATP analogs with peptide fragments of tubulin or with tubulin dimers. Kcm1, a motor protein homolog that is a machine for breaking up microtubules while attached to kinetochores is the disassembly machine. It is interesting because it binds to and stabilizes tubulin dimers. These X-ray structures are important for understanding of the mechanisms of motility. Including tubulin or peptide fragments in the complexes is essential because4 the conformational changes that kinesins use for motility are generated by its interactions with ATP and tubulin. KCM1, the new protein targeted for structural studies, is a member of the group of proteins that are catastrophe factors. KCM1 is distinct from kinesins in having a central motor domain in the polypeptide sequence with a short coiled coil stalk at the C terminus and an N-terminal globular domain that binds to kinetochores of chromosomes. Our strategy is to obtain pure domains of NCD, kinesin and KCM1 and to express these in E. coli. The purified proteins will be assayed for ATPase activity and for binding to tubulin dimers. Tubulin will be prepared from avian erythrocytes as this sample is relatively isogenic and homogeneous. We will purify the complexes and crystallize them for X-ray diffraction studies.