| Budget Amount *help |
¥11,300,000 (Direct Cost: ¥11,300,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Research Abstract |
F1-ATPase, with a subunit composition of α3β3γδε, is a catalytic sector of the membrane bound ATP synthase. The rotational catalysis mechanism of F1 is accompanied by rotation of the rod-like g subunit. We previously solved the structure of the a3b3 sub-assembly of F1-ATPase from a thermophilic bacterium Bacillus PS3. We have extended the structural study to nuceotide-bound a3b3 subassembly and α3β3γ and α3β3γε sub-assemblies.
We looked at the structure of the nucleotide-bound form of the α3β3 sub-assembly by analyzing x-ray diffraction data from the nucleotide-soaked crystals. We could trap a state where the over-all structure is very similar to that of the nucleotide-free state, but where phosphate binding to the β-subunit gets weaker than in the nucleotide-free state. Also we have tried to crystallize the sub-assembly in presence of non-hydrolyzable nucleotide analog AMPPNP, and got crystals.
We have made considerable efforts to get good crystals of the α3β3γ sub-assembly. Both preparation method and crystallization conditions have been examined extensively. We found that those crystals diffracted to resolutions of 15-10A at SPring8. Although the values were poor, they were twice better than values for resolution limit obtained with laboratory x-ray source. Further efforts are being made to extend the resolution of the crystals and to find appropriate conditions for cooling crystals.
The α3β3γε sub-assembly crystals have been found to be much better than the α3β3γ sub-assembly crystals described above. With SPring8 beam, we collected a data set to resolution of 4.5 A at 100K.The unit cell parameters were a=225.3A, b=225.7A, c=224.6A, α=94.1, β=117.5, γ=117.8, and the unit cell contains 8 of the sub-assembly. The data were merged with Rmerge of 12.2% at 4.5 A resolution. Structural analysis by molecular replacement is in progress.
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