Tokuraku Kiyotaka, Kurogi Rika, Toya Ryo, Uyeda Taro Q P
Department of Chemical Science and Engineering, Miyakonojo National College of Technology, 473-1 Yoshio-cho, Miyakonojo-shi, Miyazaki 885-8567, Japan.
J Mol Biol. 2009 Feb 13;386(1):149-62. doi: 10.1016/j.jmb.2008.12.008. Epub 2008 Dec 11.
Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca(2+)-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg(2+)-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg(2+)-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.
通过多种不同方法已检测到肌球蛋白与肌动蛋白丝之间的协同相互作用，并且有人提出这种相互作用在肌动球蛋白马达产生力的过程中发挥一定作用。在本研究中，我们直接利用荧光显微镜观察肌球蛋白与肌动蛋白丝的结合情况，以更详细地分析协同相互作用的机制。为此，我们制备了兔骨骼肌肌球蛋白和盘基网柄菌肌球蛋白II的荧光标记重酶解肌球蛋白（HMM）。当以亚化学计量添加时，这两种类型的HMM均沿肌动蛋白丝形成荧光簇。对HMM簇荧光强度的定量分析表明存在两种不同类型的协同结合。在结合亚化学计量鬼笔环肽的Ca(2+) - 肌动蛋白丝上观察到较强的形式，其中簇中HMM分子的密度与完全标记时相当。在有和没有化学计量鬼笔环肽的Mg(2+) - 肌动蛋白丝上观察到新的、较弱的形式。较弱形式簇中的HMM密度比完全标记时低几倍。弱协同结合需要亚微摩尔ATP，在没有核苷酸或存在ADP和ADP - Vi时不会发生。盘基网柄菌HMM的G680V突变体在存在肌动蛋白丝和ATP的情况下过度占据ADP - Pi结合状态，它也沿Mg(2+) - 肌动蛋白丝形成簇，这表明HMM与肌动蛋白丝的弱协同结合发生或起始于肌动球蛋白 - ADP - Pi复合物的中间状态，而不是通过添加ADP - Vi所达到的状态。
