Mechanochechemical Coupling in Brain Myosin V

肌球蛋白V的持续合成能力：我们证明了肌球蛋白V的单个分子沿着附着在基质上的肌动蛋白轨道向前移动。本研究项目就是基于这一发现而开发的。肌球蛋白V进行性运动的分析：获得相邻步进位移之间的停留时间对ATP浓度的依赖性。假设两个连续的反应步骤，分析数据的直方图，这导致k_1/[ATP]= 0.4s ^<-1>M^<-1>，k_2 =18 s^<-1>。k_2值远大于ATP酶速率，为1.2s^<-1>/头。因此，这两个连续反应不能解释为ADP释放步骤和随后的ATP结合步骤。单头肌球蛋白V的制备和表征：我们发现在Ca^&lt;2+&gt;存在的情况下，用蛋白酶K消化肌球蛋白V能够制备单头肌球蛋白V（S1），然后发现S1不是进行性的。从S1重建双头HMM：通过linki重建HMM ...更多信息 ng两个S1，带有两个柔性PEG链。其中一个头部用荧光团标记，然后观察其沿沿着肌动蛋白丝的运动。我们发现这种隐马尔可夫模型是渐进的，其步长约为70 nm。通过柔性PEG链连接的两个头部不相互机械约束。在摆动前臂模型中，头部的弯曲被认为是使尾部头部与肌动蛋白分离并向前移动。因此，该模型不能解释该HMM的移动。此外，步长（70 nm）远小于最大跨度（即，2x（70+36）nm），与肌动蛋白丝的螺距大致一致。HMM纳米级动力学行为的高速AFM成像：使用高速AFM，我们在从笼状ATP释放ATP之前和之后对肌球蛋白V进行成像。结果发现，肌球蛋白V释放ATP后，头部迅速弯曲，并在1-2s内恢复到原来的构象。对于肌动蛋白-肌球蛋白V，我们发现发生了以下事件：肌球蛋白V的一个头部附着在肌动蛋白丝上后不久，头部就沿着肌动蛋白丝脱位了。这种行为不能用摆动的双臂模型来解释。无生命物体（珠子）的运动：当珠子和肌球蛋白V都存在时，珠子沿着肌动蛋白轨迹向前运动。这个观察结果不能用一个模型来解释，这个模型假设头部向前移动时从肌动蛋白丝上脱离，强烈地表明头部向前移动，与肌动蛋白丝保持接触。少

Processivity of Myosin V : We evidenced that single molecules of myosin V move processively along actin tracks attached to a substratum. The present research project was developed based on this finding. Analysis of Myosin V Processive Movement : Dependence on the ATP concentration of the dwell time between adjacent step-wise displacements was obtained. The histogram of the data was analyzed assuming two continuous reaction steps, which resulted in k_1/[ATP]=0.4 s^<-1>M^<-1>,k_2=18 s^<-1>. The value of k_2 is much larger than the ATPase rate, 1.2s^<-1>/head. Therefore, the two continuous reactions cannot be interpreted as the ADP-release step and the subsequent ATP-binding step. Preparation and Characterization of Single-headed Myosin V : We found that single-headed myosin V(S1) was able to be prepared by digesting myosin V with proteinase K in the presence of Ca^<2+>, and then found that S1 was not processive. Reconstruction of Double-headed HMM from S1 : HMM was reconstructed by linki … More ng two S1s with two flexible PEG chains. One of the head was labeled with a fluorophore, and then its movement along actin filaments was observed. We found that this HMM was processive, and its step size was about 70nm. The two heads linked via flexible PEG chains do not mechanically constrain each other. In the swinging leverarm model, bending of the leading head is supposed to detach the trailing head from actin and bring it forward. Therefore, the movement of this HMM cannot be accounted for by this model. In addition, the step size(70nm) is much smaller than the maximum span(i.e.,2x(70+36)nm), and approximately coincides with the helical pitch of an actin filament. High-speed AFM imaging of nanometerscale dynamic behavior of HMM : Using a high-speed AFM we imaged myosin V before and after releasing ATP from caged-ATP. We found that the head of myosin V bent quickly soon after releasing ATP, and returned to the original conformation in 1-2s. With acto-myosin V we found following events to take place ; soon after one head of myosin V was attached to an actin filament, the head dislocated along the actin filament. This behavior cannot be explained by the swinging leverarm model. Movement of an inanimate object(bead) : When both beads and myosin V were present, the beads moved processively along actin tracks. This observation cannot be accounted for by a model that assumes detachment from an actin filament of the head that is moving forward, and strongly suggests that the head is moving forward, keeping in contact with an actin filament. Less