Mechanochechemical Coupling in Brain Myosin V

肌凝蛋白V的前进性：我们证明了肌凝蛋白V的单分子沿着附着在基质上的肌动蛋白轨道前进。目前的研究项目就是基于这一发现而展开的。肌凝蛋白V的运动分析：获得了相邻位移之间停留时间与ATP浓度的关系。采用两个连续反应步骤对数据进行直方图分析，得到k_1/[ATP]=0.4 s^<-1>M^<-1>,k_2=18 s^<-1>。k_2的值远远大于atp酶速率，为1.2s^<-1>/头。因此，这两个连续反应不能被解释为adp释放步骤和随后的atp结合步骤。单头肌凝蛋白V的制备与表征：我们发现在Ca^<2+>存在的情况下，用蛋白酶K消化肌凝蛋白V可以制备单头肌凝蛋白V(S1)，然后发现S1不具有进程性。将S1: HMM与两个柔性PEG链连接，重建了双头HMM。其中一个头部用荧光团标记，然后观察其沿肌动蛋白丝的运动。我们发现该HMM是渐进的，其步长约为70nm。通过柔性PEG链连接的两个头不会机械地相互约束。在摆动杠杆模型中，通过前导头的弯曲将后导头与肌动蛋白分离，使其向前移动。因此，该模型不能解释HMM的运动。此外，步长（70nm）远小于最大跨度(即。，2x(70+36)nm)，与肌动蛋白丝的螺旋节距大致重合。高速AFM成像HMM纳米尺度动态行为：使用高速AFM成像肌凝蛋白V从笼状ATP释放ATP前后。我们发现肌凝蛋白V的头部在释放ATP后迅速弯曲，并在1-2s内恢复到原来的构象。对于肌动蛋白V，我们发现发生了以下事件；肌凝蛋白V的一个头部附着在肌动蛋白丝上后不久，头部沿着肌动蛋白丝脱位。这种行为不能用摆动杠杆模型来解释。无生命物体的运动：当珠状体和肌凝蛋白V同时存在时，珠状体沿着肌动蛋白轨迹运动。这种观察结果不能用一个模型来解释，该模型假设头部与向前移动的肌动蛋白丝分离，并强烈表明头部正在向前移动，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