Mechanochechemical Coupling in Brain Myosin V

肌球蛋白V的加工性：我们证明了肌球蛋白V的单分子沿着附着在底物上的肌动蛋白轨迹进行连续运动。本研究项目就是在这一发现的基础上展开的。肌球蛋白V进行性运动分析：获得了相邻步进位移之间的停留时间对ATP浓度的依赖关系。数据的直方图分析假定有两个连续的反应步骤，得到k1/[ATP]=0.4 S^-1；M^&lt；-1&gt；，k2=18 S^&lt；-1&gt；K_2值远大于ATPase速率，为1.2s^~(-1)/头。因此，这两个连续的反应不能解释为ADP释放步骤和随后的ATP结合步骤。单头肌球蛋白V的制备及性质：我们发现，单头肌球蛋白V(S1)是在Ca^&lt；2+&gt；存在的情况下，用蛋白酶K消化得到的，且S1不是进行性的。基于LINKI…的双头隐马尔可夫模型重构更多的NG两个S1，带有两个灵活的聚乙二醇链。其中一个头部被标记了荧光团，然后观察其沿肌动蛋白细丝的运动。我们发现这种隐马尔可夫模型是进行性的，其步长约为70 nm。通过柔性的聚乙二醇链连接的两个头不会机械地相互约束。在摆动杠杆臂模型中，前头的弯曲被认为是将后头从肌动蛋白中分离出来，并将其向前推进。因此，这种隐马尔可夫模型的运动不能用这个模型来解释。此外，步长(70 Nm)远小于最大跨度(即2x(70+36)nm)，并与肌动蛋白细丝的螺旋间距大致一致。HMM纳米级动态行为的高速原子力显微镜成像：使用高速原子力显微镜对笼养的三磷酸腺苷释放三磷酸腺苷前后肌球蛋白V进行成像。我们发现，肌球蛋白V的头部在释放ATP后不久迅速弯曲，并在1-2s内恢复到原来的构象。对于acto-myosin V，我们发现发生了以下事件：在肌球蛋白V的一个头部连接到肌动蛋白细丝后不久，头部沿着肌动蛋白细丝脱位。这种行为不能用摆动的杠杆模型来解释。无生命物体(珠)的运动：当珠和肌球蛋白V同时存在时，珠沿着肌动蛋白轨迹进行移动。这种观察结果不能用假设脱离头部向前移动的肌动蛋白细丝的模型来解释，并强烈表明头部正在向前移动，保持与肌动蛋白细丝的接触。较少

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