Noguchi Taro Q P, Morimatsu Masatoshi, Iwane Atsuko H, Yanagida Toshio, Uyeda Taro Q P
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan; Department of Chemical Science and Engineering, National Institute of Technology, Miyakonojo College, Miyakonojo, Miyazaki, Japan.
Nanobiology Laboratories, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
PLoS One. 2015 May 6;10(5):e0126262. doi: 10.1371/journal.pone.0126262. eCollection 2015.
The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.
肌动蛋白的结构动力学，包括小结构域和大结构域之间的倾斜运动，对于与肌动蛋白结合蛋白的正常相互作用至关重要。Gly146位于两个结构域之间的铰链处，我们之前表明G146V突变会导致骨骼肌肌球蛋白出现严重的运动缺陷，但对肌球蛋白V的运动没有影响。本研究检验了这样一个假设，即G146V突变会损害两个结构域之间的旋转，从而导致这种功能缺陷。首先，我们的研究表明，G146V细丝的解聚比野生型细丝慢。考虑到最近关于肌动蛋白细丝稳定涉及两个结构域旋转的报道，这一结果与G146V细丝与野生型细丝结构状态的差异是一致的。接下来，我们在有或没有骨骼肌重酶解肌球蛋白或肌球蛋白V的重酶解肌球蛋白等效物存在且有ATP的情况下，测量了两个结构域中两个荧光团之间的分子内荧光共振能量转移（FRET）效率。单分子FRET测量表明，在存在骨骼肌重酶解肌球蛋白的情况下，对照和G146V肌动蛋白细丝的肌动蛋白亚基构象不同。G146V亚基这种改变的构象可能导致肌球蛋白II出现运动缺陷。相比之下，在存在肌球蛋白V的情况下，对照和G146V亚基的FRET效率分布相似，这与G146V肌动蛋白与肌球蛋白V缺乏运动缺陷一致。在存在肌球蛋白V的情况下FRET效率的分布与在存在骨骼肌重酶解肌球蛋白的情况下不同，这意味着肌动蛋白构象在肌球蛋白运动中的作用取决于肌球蛋白的类型。
