| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2004: ¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 2003: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Research Abstract |
We have succeeded to express recombinant fragment of Dictyostelium cytoplasmic dynein in Dictyostelium cells (Nishiura et al. J.Biol.Chem (2004)). The expressed dynein fragment with molecular mass of 380kDa is consisted of the C-terminal heavy chain fragment, and maintains full motor activities : the fragment, a single-headed dynein motor, has high microtubule-activated ATPase activity (120 s^<-1>), and drives microtubule sliding at 3 μm/sec. By microtubule-landing assays, the sliding is shown to be non-processive, and the duty ratio is determined as 0.37. By combining these values, the step size is expected to be 8 nm. Thus, this active recombinant cytoplasmic dynein fragment behaves like the single-headed kinesin, another microtubule-based motor. By using this unique expression system, we have examined functional roles of four potential ATPase sites in the AAA+-ring of the dynein motor domain. We introduced point mutations in the highly conserved Walker A and Walker B sequences in these four potential ATPase sites, and examined their basal and microtubule-activated ATPase activities as well as their motility. We have found that the first AAA+ module has the primary ATPase site responsible for the force generation, and the third AAA+ module has the ATPase site tightly coupled with the former (Kon et al., Biochemistry (2004)). We have then developed the GFP-based FRET system to detect ATP hydrolysis cycle-dependent structural transitions of dynein, which may correspond to power stroke (Kon et al., Nature Structural and Molecular Biology (2005)). We identified two distinct structural states that have distinct fluorescence spectra due to FRET between GFP and BFP attached to two locations on the active dynein motor. We also correlated these two states to intermediate states of cyclic ATP hydrolysis at the dynein primary ATPase site.
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