Sheng Sitong, Gao Yan, Khromov Alexander S, Somlyo Avril V, Somlyo Andrew P, Shao Zhifeng
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0736, USA.
J Biol Chem. 2003 Oct 10;278(41):39892-6. doi: 10.1074/jbc.M306094200. Epub 2003 Aug 6.
The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.
本研究的目的是确定天然双头肌球蛋白的一个头部被另一个头部进行空间位阻，是否是导致非磷酸化双头肌球蛋白与单头肌球蛋白的高活性相比无活性的原因，这是基于重酶解肌球蛋白二维晶体的电子显微镜观察结果所提出的（温特，T.，泰勒，D.，梅西耶，T.，特赖布斯，K.M.，以及泰勒，K.A.（1999年）《细胞生物学杂志》147卷，1385 - 1390页；以及温特，T.，泰勒，D.，特赖布斯，K.M.，和泰勒，K.（2001年）《美国国家科学院院刊》98卷，4361 - 4366页）。我们早期的低温原子力显微镜（cryo - AFM）（张，Y.，邵，Z.，索姆利奥，A.P.，和索姆利奥，A.V.（1997年）《生物物理学杂志》72卷，1308 - 1318页）表明，调节轻链的硫代磷酸化增加了单个肌球蛋白分子两个头部之间的间距，但两个头部之间通过强结合产生空间位阻的热力学概率尚未确定。我们现在报告通过对显示具有正常低ATP酶活性（0.007 s⁻¹）的单个完整肌球蛋白分子进行低温原子力显微镜测定得到的这个概率。我们发现，与紧密相邻的头部相比，非磷酸化肌球蛋白相对头部位置的热力学概率在分离的头部之间大致相等（能量差为0.24 kT（其中k是玻尔兹曼常数，T是绝对温度）），并且硫代磷酸化增加了具有分离头部的分子数量（能量优势为 - 1.2 kT（其中k是玻尔兹曼常数，T是绝对温度））。我们的结果不支持这样的观点，即一个头部与另一个头部的强结合稳定了受阻构象以抵抗热涨落，从而导致空间位阻，这可以解释非磷酸化双头肌球蛋白的低活性。
