The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation
The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation
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DOI：
10.1529/biophysj.105.079087
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发表时间：
2006-06-01
影响因子：
3.4
通讯作者：
Kinosita, Kazuhiko, Jr.
中科院分区：
文献类型：
--
作者：
Hossain, Mohammad Delawar;Furuike, Shou;Kinosita, Kazuhiko, Jr.
F-1-ATP酶是ATP驱动的旋转分子马达，其中中心γ亚基在由α（3）β（3）亚基组成的定子圆柱体内旋转。为了阐明转子-定子相互作用在扭矩产生中的作用，我们截短了其羧基末端的γ亚基，这形成了深入定子圆柱体的a螺旋。我们使用来自嗜热芽孢杆菌PS3的F-1-ATP酶的α（3）β（3）γ亚复合物，并在大肠杆菌中表达。我们可以获得纯化的亚复合物，其中14，17或21个氨基酸残基被删除。通过将0.49 μ m珠的双链体连接到γ-亚基上来监测截短突变体的旋转特征，在20 nM-2 mM的ATP浓度下，与野生型的旋转特征没有很大差异，最显著的效果是：扭矩减少50%，缺失21个残基后ATP结合率减少70%。在散装样品中估计的ATP水解活性受到更严重的影响。特别地，21-缺失突变体的活性低> 10倍，但这可能是由于该亚复合物的不稳定性。对于扭矩产生，虽然不是为了快速催化，但在γ亚单元的穿透部分的较深的一半上的大多数转子-定子接触是不连续的。
F-1-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an a helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F-1-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-mu m beads to the gamma-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM-2 mM, the most conspicuous effect being; 50% reduction in torque and; 70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was > 10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the gamma-subunit are dispensable.