Smyczynski C, Kasprzak A A
Centre de Recherches de Biochimie Macromoléculaire, CNRS, BP 5051, 34033 Montpellier Cedex, France.
Biochemistry. 1997 Oct 28;36(43):13201-7. doi: 10.1021/bi970746i.
The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to produce approximately 11 nm steps. To probe the spatial position of the putative lever in various S1 states, we have measured, by fluorescence resonance energy transfer (FRET), the effect of nucleotides and actin on the distances between Cys-177 of the essential light chain A1 (which is attached to the alpha-helix) and three loci in the catalytic domain. Cys-177 (donor) was labeled with 1,5-IAEDANS. The trinitrophenylated ADP analog (TNP-ADP, acceptor) was used to measure the distance to the active site. Lys-553 at the actin-binding site, labeled with a fluorescein derivative, and Lys-83 modified with trinitrobenzenesulfonic acid served as two other acceptors. FRET measurements were performed for S1 alone, for its complexes with MgADP and MgATP, for the analogs of the transition state of the ATPase reaction, S1.ADP.BeFx, S1.ADP.AlF4, and S1.ADP.VO4, and for acto-S1 in the absence and in the presence of ADP. When the transition state and acto-S1 complexes were formed, the change in the Cys-177 --> Lys-83 distance was <1.1 A, for the distance Cys-177 --> Lys-553, the change was +/-2.5 A. These distance changes correspond to rotations by <10 degrees and approximately 25 degrees, respectively. For the Cys-177 --> TNP-ADP the interprobe separation decreased by approximately 6 A in the presence of BeFx and AlF4- but only 1.9 A in the presence of vanadate; we do not interpret the 6 A change as resulting from the lever rotation. Using the coordinates of the acto-S1 complex, we have computed the expected changes in these distances resulting from rotation of the lever. These changes were much greater than the ones observed. The above results are inconsistent with models of force generation by S1 in which the head assumes two distinct conformations characterized by large differences in the angle between the motor and the light chain-binding domain. Several alternative mechanisms are proposed.
肌球蛋白头部（S1）的X射线结构显示存在一种长α螺旋结构，该结构支撑必需轻链和调节轻链。有人提出，S1催化结构域中的微小结构变化通过摆动长α螺旋（“杠杆臂”）得以放大，从而产生约11纳米的步幅。为了探究假定杠杆在各种S1状态下的空间位置，我们通过荧光共振能量转移（FRET）测量了核苷酸和肌动蛋白对必需轻链A1的半胱氨酸-177（连接到α螺旋）与催化结构域中三个位点之间距离的影响。半胱氨酸-177（供体）用1,5-IAEDANS标记。三硝基苯基化的ADP类似物（TNP-ADP，受体）用于测量到活性位点的距离。肌动蛋白结合位点的赖氨酸-553用荧光素衍生物标记，用三硝基苯磺酸修饰的赖氨酸-83用作另外两个受体。对单独的S1、其与MgADP和MgATP的复合物、ATP酶反应过渡态的类似物S1.ADP.BeFx、S1.ADP.AlF4和S1.ADP.VO4以及在有无ADP情况下的肌动蛋白-S1进行了FRET测量。当形成过渡态和肌动蛋白-S1复合物时，半胱氨酸-177→赖氨酸-83距离的变化小于1.1埃，对于半胱氨酸-177→赖氨酸-553距离，变化为±2.5埃。这些距离变化分别对应于小于10度和约25度的旋转。对于半胱氨酸-177→TNP-ADP，在存在BeFx和AlF4-的情况下，探针间距离减少约6埃，但在存在钒酸盐的情况下仅减少1.9埃；我们不认为6埃的变化是由杠杆旋转引起的。利用肌动蛋白-S1复合物的坐标，我们计算了杠杆旋转导致的这些距离的预期变化。这些变化远大于观察到的变化。上述结果与S1产生力的模型不一致，在该模型中，头部呈现两种不同的构象，其特征在于马达与轻链结合结构域之间的角度存在很大差异。提出了几种替代机制。
