Orientational changes of crossbridges during single turnover of ATP
Orientational changes of crossbridges during single turnover of ATP
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DOI：
10.1016/s0006-3495(03)75049-8
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发表时间：
2003-04-01
影响因子：
3.4
通讯作者：
Akopova, I
中科院分区：
文献类型：
--
作者：
Borejdo, J;Akopova, I
肌动蛋白结合的肌球蛋白横桥的旋转导致肌肉收缩。横桥由球状N-末端催化结构域和α-螺旋C-末端调节结构域组成，含有必需和调节轻链。必需轻链以两种同种型存在，其中较大的一种在N末端添加了41个氨基酸的延伸片段。催化结构域负责与肌动蛋白结合，并为产生主要作用力的事件（调节结构域的“摆动”）奠定基础。我们测量了与ATP的单个分子的营业额相关的摆动的动力学。通过用荧光加合物替换天然必需或调节轻链，在调节结构域标记肌肉。通过荧光的各向异性来测量旋转，所述荧光来自位于由显微镜的共焦孔径限定的小体积中的类似于400个横桥。通过化学计量量的ATP的快速光生作用使交叉桥同步化。旋转反映了细丝的解离，然后是缓慢的重新附着。每个轻链的解离是相同的（半衰期类似于120 ms），但重新附着的速率取决于轻链的类型。必需轻链亚型1和3的半衰期分别为920 +/- 50 ms和660 +/- 100 ms。寿命如此之长的原因是产生了少量的ATP，仅够一次跨桥周转。构建了一个模型，量化了这种影响。考虑到减速后，解离和附着的半衰期分别为34和200 ms。
Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the alpha-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added at the N-terminus. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. We measured the kinetics of the swing associated with the turnover of a single molecule of ATP. Muscle was labeled at the regulatory domain by replacing native essential or regulatory light chain with fluorescent adducts. The rotations were measured by the anisotropy of fluorescence originating from similar to400 crossbridges residing in a small volume defined by a confocal aperture of a microscope. The crossbridges; were synchronized by rapid photogeneration of a stoichiometric amount of ATP. The rotations reflected dissociation from thin filaments followed by a slow reattachment. The dissociation was the same for each light chain (halftime similar to120 ms) but the rate of reattachment depended on the type of light chain. The halftimes were 920 +/- 50 ms and 660 +/- 100 ms for isoforms 1 and 3 of the essential light chain, respectively. The reason that the lifetimes were so long was creation of a small amount of ATP, enough only for a single turnover of crossbridges. A model was constructed that quantitized this effect. After accounting for the slowdown, the halftimes of dissociation and attachment were 34 and 200 ms, respectively.