The long-term goal of this proposal is to establish the structural and mechanistic basis for force production by biological motors in general and the microtubule-kinesin system specifically. Of the three known classes of eukaryotic motors, kinesin is the smallest and most simple in terms of structural complexity. Previous studies defined the three-dimensional crystal structure of dimeric kinesin and defined the essential features of its microtubule-activated ATPase activity. The proposal has five specific aims: 1) Perform a complete kinetic analysis to establish the pathway and rate constants for rat brain kinesin using stopped-flow and chemical quench-flow methods. 2) Examine the kinetics of kinesin-microtubule binding and release from intermediate states with one head bound using stopped-flow fluorescence methods including fluorescence energy transfer. 3) Analyze the communication between the nucleotide-binding site and microtubule-binding site using a combination of site-directed mutagenesis and kinetic analysis. 4) Analyze the communication between subunits and identify residues responsible for transmission of the nucleotide-binding state to the dimer interface using site-directed mutagenesis and detailed kinetic analysis. 5) Establish the solution structure and dynamics of the coiled-coil neck domain by NMR to determine how much it may open and close during each ATPase cycle using NMR methods. These goals will be addressed by a compreheensive kinetic analysis of the step in the pathway, structural studies by NMR to define the molecular basis for force production and motility studies by light microscopy. 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.
| 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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