THE CONFORMATIONAL STATE OF THE ACTO-S-1 COMPLEX

我们提出，在肌动球蛋白ATPase周期中，肌球蛋白 跨桥在两种不同的主要构象之间交替 它们与肌动蛋白的结合强度以及它们的整体 结构。在一种构象中，在缺乏ATP的情况下，肌球蛋白 与肌动蛋白以45度角紧密结合。在第二个 构象，只有当ATP或ADP.PI结合到 肌球蛋白，肌球蛋白与肌动蛋白以90度角弱结合。 学位。我们现在已经获得了两个不同的 使用交联化的肌动蛋白构象状态S-1。两者都是负数 通过染色和冷冻蚀刻技术，电子显微照片 交联型放线菌.S-1显示了交联型的整体结构 Actin.S-1在ATP存在下非常无序，个体 以不同角度居中连接肌动蛋白的交联S-1分子 90度。在没有ATP的情况下，交联的肌动蛋白S-1显示 典型的箭头外观，特点为45度 构象。还研究了交联物的结构。 在存在ATP类似物AMP-PNP的情况下。与ATP不同的是， 交联型放线菌素S-1的结构在存在时表现得非常严谨 提示三磷酸腺苷引起的结构变化 具体的。除了这些结构研究外，我们还获得了 生物化学证据表明，交联型Actin.S-1可以存在于两个不同的 通过研究肌钙蛋白原肌球蛋白对血管内皮细胞构象的影响 交联丝。我们发现，交联物的构象 肌动蛋白。S-1依赖于与S-1结合的核苷酸。另一方面， 交联型actin.pPDM.S-1和交联型actin.NEM.S-1始终保持在 90度弱结合构象和45度强结合构象， 分别与S-1结合的核苷酸无关。这是在 与我们之前的研究一致，这些研究表明，无论 与S-1、pPDM.S-1和NEM.S-1结合的核苷酸形成稳定的肌动蛋白 分别是90度和45度构象的类似物。

We have proposed that during the actomyosin ATPase cycle, the myosin cross-bridge alternates between two major conformations, which differ markedly in their strength of binding to actin and in their overall structure. In one conformation, which occurs in the absence of ATP, myosin binds very tightly to actin at a 45 degree angle. In the second conformation, which occurs only transiently when ATP or ADP.Pi is bound to myosin, myosin binds weakly to actin at an angle postulated to be 90 degrees. We have now obtained structural evidence for the two different conformational states by using cross-linked actin.S-1. Both by negative staining and by freeze etching techniques, electron micrographs of cross-linked actin.S-1 shows that the overall structure of the cross-linked actin.S-1 is very disordered in the presence of ATP, with individual cross-linked S-1 molecules attaching to actin at variable angles centering on 90 degrees. In the absence of ATP, the cross-linked actin.S-1 shows the typical arrowhead appearance, characteristic of the 45 degrees conformation. The structure of the cross-linked complex was also examined in the presence of the ATP analog, AMP-PNP. In contrast to ATP, the structure of cross-linked actin.S-1 appears very rigor-like in the presence of AMP-PNP suggesting that the structural change induced by ATP is specific. In addition to these structural studies, we have also obtained biochemical evidence that cross-linked actin.S-1 can exist in two different conformations by studying the effect of troponin-tropomyosin on the cross-linked filament. We found that the conformation of cross-linked actin.S-1 depends on the nucleotide bound to the S-1. On the other hand, cross linked actin.pPDM.S-1 and cross-linked actin.NEM.S-1 always remain in the 90 degrees weak-binding and 45 degrees strong-binding conformations, respectively, regardless of the nucleotide bound to S-1. This is in agreement with our previous studies which show that, irrespective of the nucleotide bound to S-1, pPDM.S-1 and NEM.S-1 form with actin stable analogs of the 90 degrees and 45 degrees conformations, respectively.