Single molecule energetics of F1-ATPase motor
Single molecule energetics of F1-ATPase motor
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DOI：
10.1529/biophysj.106.097170
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发表时间：
2007-03-01
影响因子：
3.4
通讯作者：
Noji, Hiroyuki
中科院分区：
文献类型：
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作者：
Muneyuki, Eiro;Watanabe-Nakayama, Takahiro;Noji, Hiroyuki
运动蛋白在生命过程中是必不可少的，因为它们将ATP水解的自由能转化为机械功。然而，当ATP水解后释放不同数量的自由能时，它们是如何工作的基本问题仍然没有答案。为了回答这个问题，有必要澄清运动蛋白的步进运动如何在单分子水平上反映其单个动作中的ATP， ADP和P-i的浓度。ATP合成酶的F部分，也被称为F-1-ATP酶，是一个旋转分子马达，其中中心的γ亚基围绕α (3) β(3)圆柱体旋转。在低ATP浓度下，电机表现出清晰的阶跃运动。该电机的旋转动作是渐进的，并产生高扭矩。这些特性对于探索自由能输入与机械功输出之间的关系是理想的，但存在一个严重的问题，即该电机受到ADP的严重抑制。在这项研究中，我们通过引入几个突变来克服ADP抑制的问题，同时保持高酶活性。通过改变ADP的浓度，利用附着珠的探针，在大范围的自由能值下检测了对粘性载荷的步进旋转。结果表明，ATP水解的自由能不影响各步运动的表观功，但各步运动的频率与自由能有关。这是第一次在单分子水平上检测分子马达的步进运动，同时系统控制G（ATP）。结果表明，单分子水平上微观定义的功不能与宏观定义的自由能输入直接比较。
Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P-i in its individual actions at a single molecule level. The F, portion of ATP synthase, also called F-1-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of Delta G(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.