The long term aims of this proposal are to establish the structural and mechanistic basis for force production by biological motors in general and the microtubule-based ATPases kinesin and ncd specifically. Kinesin is a microtubule dependent motor responsible for the intracellular movements of membranous organelles in axons. It is the first characterized protein of a class of kinesin-related ATPases implicated ina wide variety of biological processes. The members of the kinesin superfamily all share significant sequence homology within the kinesin motor domain, and each kinesin-related protein contains short segments of extremely high homology within the ATP binding pocket, the microtubule binding domain as well as well as t=other areas not yet defined functionally. In addition, each kinesin-related protein contains a nonmotor domain that is unique and is believed to confer biological specificity to the motor. Ncd is a kinesin- related protein involved in female meiosis and embryonic mitosis. It is an interesting motor to study in comparison to kinesin because there is 40-45% sequence identity within about 350 amino acids of the motor domain which implies that the proteins will have very similar three-dimensional structures. Yet as motors, the two ATPases are distinctively different. Ncd promotes microtubule translocations in the opposite direction of kinesin, translocations are significantly slower than kinesin's, and the experiments to date suggest that ncd is not a processive ATPase as is kinesin. The goal of the research proposed here is to understand the mechanistic basis of these differences.
The specific aims are 1) to measure the kinetics of each step in the reaction pathway of the microtubule-ncd ATPase, 2) to determine whether the ncd ATPase is processive or distributive in its interactions with the microtubule. To accomplish these goals will require a detailed and comprehensive kinetic and thermodynamic investigation using presteady state kinetic techniques including rapid quench and stopped-flow approaches. It is only by this direct measurement of event at the active site of the enzyme can the mechanistic information be obtained to understand the differences in energy transduction by the ncd and kinesin ATPases.
| Albracht, Clayton D; Guzik-Lendrum, Stephanie; Rayment, Ivan et al. (2016) Heterodimerization of Kinesin-2 KIF3AB Modulates Entry into the Processive Run. J Biol Chem 291:23248-23256 |
| Guzik-Lendrum, Stephanie; Rank, Katherine C; Bensel, Brandon M et al. (2015) Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules. Biophys J 109:1472-82 |
| Rayment, Ivan (2014) Structural insights into the assembly of a monomeric class V myosin. Proc Natl Acad Sci U S A 111:4351-2 |
| Cope, Julia; Rank, Katherine C; Gilbert, Susan P et al. (2013) Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules. PLoS One 8:e53792 |
| Sardar, Harjinder S; Gilbert, Susan P (2012) Microtubule capture by mitotic kinesin centromere protein E (CENP-E). J Biol Chem 287:24894-904 |
| Chen, Chun Ju; Porche, Ken; Rayment, Ivan et al. (2012) The ATPase pathway that drives the kinesin-14 Kar3Vik1 powerstroke. J Biol Chem 287:36673-82 |
| Rank, Katherine C; Chen, Chun Ju; Cope, Julia et al. (2012) Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern. J Cell Biol 197:957-70 |
| Chen, Chun Ju; Rayment, Ivan; Gilbert, Susan P (2011) Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking. J Biol Chem 286:29261-72 |
| Sardar, Harjinder S; Luczak, Vincent G; Lopez, Maria M et al. (2010) Mitotic kinesin CENP-E promotes microtubule plus-end elongation. Curr Biol 20:1648-53 |
| McIntosh, J Richard; Morphew, Mary K; Grissom, Paula M et al. (2009) Lattice structure of cytoplasmic microtubules in a cultured Mammalian cell. J Mol Biol 394:177-82 |
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