Roopnarine O, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biophys J. 1996 Jun;70(6):2795-806. doi: 10.1016/S0006-3495(96)79849-1.
We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.
我们利用电子顺磁共振研究了在核苷酸和核苷酸类似物存在的情况下肌球蛋白头部的取向，以诱导模拟肌动球蛋白ATP酶循环中间态的平衡态。我们获得了与去皮兔腰大肌纤维中肌球蛋白头部的半胱氨酸707（SH1）结合的茚二酮自旋标记（InVSL）的电子顺磁共振谱。该探针被牢固地固定在头部的催化结构域上，并且在僵直状态（无核苷酸）下探针的主轴几乎与纤维轴平行排列，使其对头部的轴向旋转直接敏感。添加ADP后，所有头部仍与肌动蛋白紧密结合，但光谱超精细分裂增加了0.55±0.02 G，对应于7度的小但显著的轴向旋转。腺苷5'-（腺苷酰亚胺二磷酸）（AMPPNP）或焦磷酸降低了肌动球蛋白的亲和力，并引入了高度无序的头部群体，类似于在松弛状态下观察到的情况。对于其余取向的群体，焦磷酸相对于僵直状态没有引起显著变化，但AMPPNP诱导了轻微但可能显著的旋转（2.2度±1.6度），方向与ADP诱导的相反。腺苷5'-O-（3-硫代三磷酸）（ATPγS）使肌肉纤维松弛，使头部与肌动蛋白完全解离，并产生与ATP松弛时类似的无序状态。ATPγS加Ca诱导了一种弱结合状态，其中大多数与肌动蛋白结合的头部无序。在ADP存在的情况下，钒酸盐的影响可忽略不计，但在等长收缩中，钒酸盐显著降低了力量和取向头部的比例。这些结果与一个模型一致，在该模型中，肌球蛋白头部在动力冲程早期无序（弱结合状态），在动力冲程后期刚性取向（强结合状态），而强结合状态之间的转变仅在催化结构域的轴向取向上引起轻微变化。
