Studies Of Myosin V

肌球蛋白V是一种双头非常规肌球蛋白，由于存在六个轻链结合IQ重复序列，因此具有延长的颈部。在细胞中，肌球蛋白V被认为是囊泡马达。小鼠肌球蛋白V的双头（HMM）和单头片段（S1），每个片段在其羧基末端标记有FLAG表位，在Sf 9细胞中与钙调蛋白一起沿着表达。此外，我们还从小鼠脑和鸡脑中纯化了肌球蛋白V。从脑中纯化的肌球蛋白V的肌动蛋白激活的MgATP酶活性被微摩尔钙的存在强烈激活，而表达的HMM即使在没有钙的情况下也很高，这表明该分子的尾部具有调节功能。多项证据表明，肌球蛋白V是一种进行性马达，可以沿着肌动蛋白丝沿着移动一定距离而不发生解离，并且两个头部可以同时结合到同一肌动蛋白丝上。两个最直接的方法来检查这是光学捕获或检查运动的单个，荧光标记的分子的肌球蛋白V的肌动蛋白丝使用全内反射显微镜。电子显微镜研究表明，绑定头之间的距离对应于13个肌动蛋白单体（36 nm）或一个螺旋重复。与这些数据一致，光学捕获结果表明，运行中步进事件之间的距离为36 nm。相比之下，运行中的第一步或单个附着事件的步长仅为25 nm。这表明，第二个头必须能够进行扩散搜索适当的肌动蛋白单体，同时被拴在所附的头。动力冲程分两个不等的步骤进行。测量附着的肌球蛋白V头的刚度在一个连续的运行过程中显示，刚度降低的时期可以检测到步进事件之前。这可能代表了短暂的单头依恋。电子显微镜下的肌球蛋白头在存在或不存在ATP不连接到肌动蛋白揭示了运动域的方向相比，杠杆臂的明显差异，这表明我们能够看到的电力中风，即使在没有肌动蛋白。 我们已经通过增加或减少IQ残基的数量来增加或减少颈区长度的突变体。野生型肌球蛋白V具有6个IQ基序。我们发现4IQ、6IQ和8IQ肌球蛋白V分子在TIRF分析和光阱中的运动是同步的。在光阱中的单个事件的步长随着颈部的长度而增加，体外运动的速率也是如此。在大多数离子条件下，2IQ肌球蛋白V不能向前移动，这表明长颈长度可能对该功能至关重要。

Myosin V is a two-headed unconventional myosin that has an extended neck due to the presence of six light chain binding IQ-repeats. In cells, myosin V is thought to be a vesicle motor. Double-headed (HMM) and single-headed fragments (S1)of mouse myosin V, each tagged with the FLAG-epitope on their carboxyl-terminal end, were expressed in Sf9 cells along with calmodulin. In addition, we purified myosin V from mouse brain and chicken brain. The actin activated MgATPase activity of myosin V purified from brain is strongly activated by the presence of micromolar calcium whereas that of expressed HMM is high even in the absence of calcium suggesting that the tail portion of the molecule has regulatory function. Several lines of evidence suggest that myosin V is a processive motor which can move some distance along actin filaments without dissociation and that the two heads can bind simultaneously to the same actin filament. The two most direct methods for examining this are optical trapping or examining the movement of single, fluorescently-labeled molecules of myosin V on actin filaments using total internal reflection microscopy. Electron microscopic studies reveal that the distance between the bound heads corresponds to 13 actin monomers (36 nm) or one helical repeat. Consistent with these data the optical trapping results show that the distance between stepping events in a run is 36 nm. In contrast, the first step in a run or the step size of a single attachment event is only 25 nm. This suggests that the second head must be able to conduct a diffusive search for the appropriate actin monomer while being tethered by the attached head. The power stroke takes place in two unequal steps. Measurement of the stiffness of the attached myosin V heads during a processive runs shows that periods of reduced stiffness can be detected just before stepping events. This probably represents transient periods of single-headed attachment. Electron microscopy of myosin heads in the presence or absence of ATP not attached to actin reveals distinctive differences in orientations of the motor domain compared to the lever arm suggesting that we're able to see the power stroke even in the absence of actin. We have made mutants that increase or decrease the length of the neck region by increasing or decreasing the number of IQ residues. The wild type myosin V has 6 IQ motifs. We find that the 4IQ, 6IQ and 8IQ myosin V molecules move processively in the TIRF assay and in the optical trap. The step size of single events in the optical trap increases with the length of the neck as does the rate of in vitro motility. Under most ionic conditions, the 2IQ myosin V is unable to move processively suggesting that the long neck length may be critical for this function.