Rebello C A, Ludescher R D
Department of Food Science, Rutgers, The State University, New Brunswick, New Jersey 08901-8520, USA.
Biochemistry. 1998 Oct 13;37(41):14529-38. doi: 10.1021/bi981240i.
To better understand the relationship between structure and molecular dynamics in F-actin, we have monitored the torsional flexibility of actin filaments as a function of the type of tightly bound divalent cation (Ca2+ or Mg2+) or nucleotide (ATP or ADP), the level of inorganic phosphate and analogues, KCl concentration, and the level of phalloidin. Torsional flexibility on the microsecond time scale was monitored by measuring the steady-state phosphorescence emission anisotropy (rFA) of the triplet probe erythrosin-5-iodoacetamide covalently bound to Cys-374 of skeletal muscle actin; extrapolations to an infinite actin concentration corrected the measured anisotropy values for the influence of variable amounts of rotationally mobile G-actin in solution. The type of tightly bound divalent cation modulated the torsional flexibility of F-actin polymerized in the presence of ATP; filaments with Mg2+ bound (rFA = 0.066) at the active site cleft were more flexible than those with Ca2+ bound (rFA = 0.083). Filaments prepared from G-actin in the presence of MgADP were more flexible (rFA = 0.051) than those polymerized with MgATP; the addition of exogenous inorganic phosphate or beryllium trifluoride to ADP filaments, however, decreased the filament flexibility (increased the anisotropy) to that seen in the presence of MgATP. While variations in KCl concentration from 0 to 150 mM did not modulate the torsional flexibility of the filament, the binding of phalloidin decreased the torsional flexibility of all filaments regardless of the type of cation or nucleotide bound at the active site. These results emphasize the dynamic malleability of the actin filament, the role of the cation-nucleotide complex in modulating the torsional flexibility, and suggest that the structural differences that have previously been seen in electron micrographs of actin filaments manifest themselves as differences in torsional flexibility of the filament.
为了更好地理解F-肌动蛋白的结构与分子动力学之间的关系，我们监测了肌动蛋白丝的扭转灵活性，它是紧密结合的二价阳离子（Ca2+或Mg2+）或核苷酸（ATP或ADP）类型、无机磷酸盐及其类似物的水平、KCl浓度以及鬼笔环肽水平的函数。通过测量与骨骼肌肌动蛋白的Cys-374共价结合的三重态探针赤藓红-5-碘乙酰胺的稳态磷光发射各向异性（rFA），监测微秒时间尺度上的扭转灵活性；外推至无限肌动蛋白浓度可校正测量的各向异性值，以消除溶液中可变数量的可旋转移动G-肌动蛋白的影响。紧密结合的二价阳离子类型调节了在ATP存在下聚合的F-肌动蛋白的扭转灵活性；在活性位点裂隙处结合Mg2+的丝（rFA = 0.066）比结合Ca2+的丝（rFA = 0.083）更灵活。在MgADP存在下由G-肌动蛋白制备的丝比用MgATP聚合的丝更灵活（rFA = 0.051）；然而，向ADP丝中添加外源无机磷酸盐或三氟化铍会使丝的灵活性降低（增加各向异性），降至MgATP存在时的水平。虽然KCl浓度从0到150 mM的变化未调节丝的扭转灵活性，但鬼笔环肽的结合降低了所有丝的扭转灵活性，无论活性位点结合的阳离子或核苷酸类型如何。这些结果强调了肌动蛋白丝的动态可塑性、阳离子-核苷酸复合物在调节扭转灵活性中的作用，并表明先前在肌动蛋白丝电子显微照片中看到的结构差异表现为丝扭转灵活性的差异。
