Investigation on the functional significance of myosin subfragment-2

我们研究了肌球蛋白亚片段2（S-2）连接的头和杆结构域的分子的功能意义。头部含有肌动蛋白结合位点和ATP水解口袋，被认为是分子机械化学功能的关键。肌球蛋白分子在杆域结合在一起，形成肌肉细胞中的粗丝。虽然一些研究表明位于头-杆连接处的S-2区在力的产生和转导中具有重要作用，但S-2区的功能作用尚未明确阐明。以鸡平滑肌肌球蛋白为模型，利用杆状病毒载体在昆虫细胞中表达重链肌球蛋白（HMM）。我们纯化了野生型和在S-2区缺失20（Δ20）和49（Δ49）个氨基酸残基的两个突变体。Δ49突变体比Δ20有更长的缺失，但保留了该区域7个氨基酸残基的周期性（七肽重复）。我们分析了分子马达功能， ...更多信息 采用体外运动试验和激光陷阱。我们测量了荧光标记的肌动蛋白丝（VEL）在固定在盖玻片上的肌球蛋白层上的滑动速度。我们还测量了在α-辅肌动蛋白存在下的速度，该辅肌动蛋白对滑动丝施加机械载荷。测量α-辅肌动蛋白浓度（ACT）作为肌球蛋白分子的平均力的指标，在该浓度下，负荷与相互作用的肌球蛋白分子产生的力平衡并且滑动运动停止。在激光陷阱实验中，确定了单位位移（Duni）、单位力（Funi）和肌动蛋白-肌球蛋白相互作用的单个事件的持续时间（Ton）。VEL和ACT在突变体（Δ20和Δ 49）中均比野生型降低。特别是，与野生型相比，Δ20的降低幅度大于Δ49，尽管缺失长度较短（VEL，-38% vs. -23%;ACT，-67% vs. -38%，分别）。无论是Duni还是Funi，在突变体和野生型之间，还是在突变体之间，都没有什么不同。值得注意的是，与野生型相比，Δ20中的Ton长26%。因此，S-2区可能在分子的运动功能中起重要作用，但不影响单个分子的步长或单位力。这也表明，7个氨基酸残基的周期性可能是重要的运动功能。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