Investigation on the functional significance of myosin subfragment-2

我们研究了连接肌球蛋白分子头部和杆状结构域的肌球蛋白亚片段2(S-2)的功能意义。头部含有肌动蛋白结合部位和ATP水解袋，被认为在分子的机械力化学功能中起关键作用。肌球蛋白分子在杆状区结合在一起，在肌肉细胞中形成一根粗大的细丝。虽然一些研究表明，位于头-杆连接处的S-2区域在力的产生和转导中发挥了重要作用，但S-2区域的功能作用尚不清楚。以鸡肌球蛋白为模型，利用杆状病毒载体在昆虫细胞中表达重链肌球蛋白(HMM)。我们纯化了S-2区20(Δ20)和49(Δ49)个氨基酸残基缺失的野生型和两个突变体。Δ49突变体比Δ20有更长的缺失，但保留了该区域的7个氨基酸残基的周期性(7个重复)。我们用…分析了分子马达的功能。更多地进行体外运动试验和激光诱捕。我们测量了荧光标记的肌动蛋白细丝(VEL)在固定在盖片上的肌球蛋白层上的滑动速度。我们还测量了在施加机械载荷的α-Actinin存在的情况下滑动细丝的速度。α-肌动蛋白浓度(ACT值)作为肌球蛋白分子平均作用力的一个指标，其负荷与相互作用的肌球蛋白分子产生的力平衡，滑动运动停止。在激光陷阱实验中，测定了肌动蛋白-肌球蛋白相互作用的单位位移(Duni)、单位作用力(Funi)和单个事件的持续时间(Ton)。突变体(Δ20和Δ49)的VEL和ACT值均低于野生型。特别是，与野生型相比，Δ20比Δ49的缺失长度更短(VEL，-38%对-23%；ACT，-67%对-38%)。突变体与野生型或突变体之间的杜尼和福尼均无差异。值得注意的是，与野生型相比，Δ20的Ton长了26%。综上所述，S-2区域可能在分子的运动功能中发挥重要作用，但不影响单个分子的步长或单位力。提示7个氨基酸残基的周期性可能对运动功能有重要影响。较少

We investigated the functional significance of myosin subfragment-2 (S-2) connecting the head and rod domains of the molecule. The head contains actin-binding site and ATP-hydrolysis pocket and is considered to be pivotal in mechanochemical function of the molecule. Myosin molecules bind together at the rod domains to form a thick filament in muscle cells. While several studies suggested significant role of the S-2 region, located in the head-rod junction, in force generation and transduction, the functional role of the S-2 region is not clearly elucidated yet. We used chicken smooth muscle myosin as a model and expressed heavy meromyosin (HMM) with baculoviral vector in cultured insect cells. We purified wild-type and two mutants that had deletions of 20(Δ20) and 49 (Δ49) amino-acid residues in the S-2 region. The Δ49 mutant had longer deletion than Δ20, but conserved the seven amino-acid residues periodicity (heptad repeat) in this region. We analyzed the molecular motor function usi … More ng the in vitro motility assay and laser trap. We measured the sliding velocity of fluorescently labeled actin filaments (VEL) over myosin layer fixed on a coverslip. We also measured the velocity in the presence of α-actinin which imposed mechanical load upon the sliding filaments. The α-actinin concentration (ACT), at which the load was balanced with the generated force of interacting myosin molecules and the sliding movement stopped, was measured as an index of the average force of myosin molecules. In laser trap experiments, the unitary displacement (Duni),the unitary force (Funi) and the duration of a single event of the actin-myosin interaction (Ton) were determined. Both VEL and ACT were decreased in the mutants (Δ20 and Δ 49) than in the wild-type. Especially, the decreases compared to the wild-type were greater in Δ20 than Δ49 albeit the shorter deletion length (VEL,-38% vs. -23%;ACT,-67% vs. -38%,respectively). Neither Duni nor Funi was different between the mutants and the wild-type or among the mutants. Notably, Ton was 26% longer in Δ20 as compared to the wild-type. In conclusion, the S-2 region may play significant roles in motor function of the molecule, but does not affect step size or unitary force of a single molecule. It is also suggested that the 7 amino-acid residue periodicity may be important for the motor function. Less