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。几条线索的证据表明，肌球蛋白V是一个进行性马达，它可以沿着肌动蛋白丝沿着移动一段距离而不解离，并且两个头部可以同时结合到同一个肌动蛋白丝上。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有六个IQ基序。我们发现，4 IQ，6 IQ和8IQ肌球蛋白V分子在TIRF测定和在大多数条件下测试的光阱中的procancelly移动，2 IQ突变体在低ATP浓度下procancelly移动。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。我们最近使用了一种新的显微镜技术，称为FIONA（荧光成像在一纳米精度），看看在步进行为的荧光标记的肌球蛋白V分子移动proc肌动蛋白。数据显示，N-末端EGFP标记的肌球蛋白V HMM，其中两个头部中只有一个被标记，需要74 nm的步长，这与手-手杠杆臂步进模型一致。用颈长突变体进行的FIONA研究表明，步长和颈长之间存在线性关系。我们已经发现，在组织纯化的肌球蛋白V的酶活性低（不含钙）的条件下，该分子折叠成紧凑的三角形形状。在通过钙或通过增加离子强度激活ATP酶活性时，分子打开以采用熟悉的T形或Y形结构。

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. 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. 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 six IQ motifs. We find that the 4IQ, 6IQ and 8IQ myosin V molecules move processively in the TIRF assay and in the optical trap under most conditions tested and that the 2IQ mutant moves processively at low ATP concentrations. 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. We have recently used a new microscopic technique termed FIONA (fluorescence imaging at one nanometer accuracy) to look at the stepping behavior of fluorescently-labeled myosin V molecules moving processively on actin. The data show that an N-terminally EGFP-tagged myosin V HMM, in which only one of the two heads are labeled, takes 74 nm steps, which is consistent with a hand-over-hand lever arm stepping model. FIONA studies with the neck length mutants show a linear relationship between the step size and the length of the neck. We have found that, under conditions in which the enzymatic activity of tissue purified myosin V is low (absence of calcium), the molecule is folded into a compact triangular shape. Upon activation of the ATPase activity by calcium or by increasing the ionic strength, the molecule opens up to adopt the familiar T- or Y-shaped structures.