Stiffness of γ subunit of F1-ATPase
Stiffness of γ subunit of F1-ATPase
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DOI：
10.1007/s00249-010-0616-9
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发表时间：
2010-11-01
影响因子：
2
通讯作者：
Noji, Hiroyuki
中科院分区：
文献类型：
--
作者：
Okuno, Daichi;Iino, Ryota;Noji, Hiroyuki
F-1-ATP酶是一种分子马达，其中γ亚基在三磷酸腺苷（ATP）水解时在α（3）β（3）环内旋转。最近对F-1-ATP酶的单分子操作的研究表明，ATP的结合速率和二磷酸腺苷（ADP）的解离速率等动力学参数强烈依赖于γ亚基的旋转角（Hirono-Hara et al. 2005; Iko et al. 2009）。这些发现提供了重要的洞察力，了解各个反应步骤如何释放能量为F-1提供动力，并且还对ATP合成以及反应步骤如何在反向旋转时逆转具有影响。关于动力学参数的角度依赖性的一个重要问题是，由于系统的扭转弹性，磁珠旋转探针的角位置可能大于γ亚基的实际位置。在本研究中，我们通过比较转子和定子之间交联前后的旋转波动，评估了来自嗜热芽孢杆菌PS3的F-1的两个不同部分的刚度：嵌入α（3）β（3）环的γ亚基的内部部分，以及γ亚基的外部部分和α（3）β（3）环（以及链霉亲和素和磁珠）的复合物。内部和其余部件的扭转刚度分别确定为约223和73 pNnm/弧度。基于这些值，估计伽马子单元的内部部分的实际角位置是在使用外部磁场停止时磁珠位置的四分之一。估计的弹性也部分地解释了F-o和F-1-ATP酶之间的内在步长失配的调节。
F-1-ATPase is a molecular motor in which the gamma subunit rotates inside the alpha(3)beta(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F-1-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the gamma subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F-1 and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the gamma subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F-1 from thermophilic Bacillus PS3: the internal part of the gamma subunit embedded in the alpha(3)beta(3) ring, and the complex of the external part of the gamma subunit and the alpha(3)beta(3) ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the gamma subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F-o and F-1-ATPase.