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-kinesin system specifically. Kinesin is a microtubule- dependent motor responsible for the intracellular movements of membrane- bound organelles. It is the father of a class of kinesin-like motor proteins implicated in a wide range of biological processes including axoplasmic transport, chromosome and nuclear movements, pigment granule translocation, and endoplasmic reticulum and Golgi membrane dynamics, and axonemal assembly and motility. Of the three known classes of eukaryotic motors, kinesin is the smallest and most simple in terms of its structural complexity. Moreover, recent work has established conditions for obtaining biochemical quantities of the kinesin motor domain by expression of a truncated Drosophila gene in E. coli. The truncated kinesin, containing 401 N-terminal amino acids and referred to as K401, retains the key features expected for a native kinesin molecule. This form of kinesin binds to microtubules with an 8 nm repeat with one kinesin head per tubulin heterodimer. Previous studies establish the essential features of the reaction pathway of the microtubule-activated ATPase. Further studies outlined in this proposal have three broad specific aims: (1) to complete the kinetic and thermodynamic description of the ATPase pathway; (2) to examine the kinetic and mechanistic basis for apparent processivity in kinesin motility and possible cooperativity between kinesin heads; and (3) to establish the structural basis for force production. These goals will be addressed by a comprehensive kinetic analysis of the ATPase pathway using transient kinetic methods, measurements of the free energy change occurring in each step in the pathway, and structural studies by electron microscopy and crystallography to define the molecular basis for force production. A comprehensive kinetic and mechanistic analysis of mutant and wild-type proteins will serve to define the relationship of the structure to energy transduction. The combination of approaches outlined here will provide rigorous and direct information to define the structural and mechanistic basis for force production by kinesin. The work will thereby provide an understanding of the biological phenomena pertaining to the role of kinesin in intracellular movement.
| Auerbach, Scott D; Johnson, Kenneth A (2005) Kinetic effects of kinesin switch I and switch II mutations. J Biol Chem 280:37061-8 |
| Auerbach, Scott D; Johnson, Kenneth A (2005) Alternating site ATPase pathway of rat conventional kinesin. J Biol Chem 280:37048-60 |
| Mandelkow, E; Johnson, K A (1998) The structural and mechanochemical cycle of kinesin. Trends Biochem Sci 23:429-33 |
| Moyer, M L; Gilbert, S P; Johnson, K A (1998) Pathway of ATP hydrolysis by monomeric and dimeric kinesin. Biochemistry 37:800-13 |
| Gilbert, S P; Moyer, M L; Johnson, K A (1998) Alternating site mechanism of the kinesin ATPase. Biochemistry 37:792-9 |
| Moyer, M L; Gilbert, S P; Johnson, K A (1996) Purification and characterization of two monomeric kinesin constructs. Biochemistry 35:6321-9 |
| Correia, J J; Gilbert, S P; Moyer, M L et al. (1995) Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341. Biochemistry 34:4898-907 |
| Gilbert, S P; Webb, M R; Brune, M et al. (1995) Pathway of processive ATP hydrolysis by kinesin. Nature 373:671-6 |
| Gilbert, S P; Johnson, K A (1994) Pre-steady-state kinetics of the microtubule-kinesin ATPase. Biochemistry 33:1951-60 |
| Gilbert, S P; Johnson, K A (1993) Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli. Biochemistry 32:4677-84 |
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