ACANTHAMOEBA MYOSIN II ROD AND MUTANTS: PHYSICAL PROPERTIES

棘阿米巴肌球蛋白II棒是一种长螺旋二聚体卷曲线圈，具有柔性铰链，含有螺旋断裂脯氨酸。先前，研究人员测定了myosin II的完整野生型棒状结构域（残基849-1509），一个铰链脯氨酸被丙氨酸取代的点突变体（P398A），以及一个整个铰链区域被删除的缺失突变体（残基384-408）在0.6和2.2 M KCl, pH 7.5条件下的热稳定性。采用分析性超离心法对肌球蛋白II野生型和铰链突变体棒的大小和形状（摩擦比）进行表征。还测定了野生型和点缺失突变体透析形成的微丝与pH 7.5下5 mM Mg （II）低离子强度缓冲液的沉降特性。此外，通过电双折射测量了肌球蛋白II棒微丝的柔韧性与Mg（II）浓度的关系；具体来说，Rau发现天然肌球蛋白II的依赖于Mg（II）的柔性到刚性的转变与野生型棒状微丝非常相似。在目前的研究中，肌球蛋白II棒c端（氨基末端694残基标记）磷酸化位点的突变使3个Asp取代3个Ser [S1489D， S1494D, S1499D] (78,615 MW)， 3个Ala取代3个Ser [S1489A， S1494A, S1499A] (78,483 MW)， 1个Asp取代Ser [S1489D] (78,559 MW)；野生型电棒功率为78,531兆瓦。B. Kunnummal （LCB）将利用野生型和突变型棒与天然肌球蛋白II制作共丝，以测试含有Ser-to-Asp取代的共聚棒是否抑制肌动蛋白激活的肌球蛋白II头部的atp酶活性（模拟这些位点的磷酸化）。如果是这样，肌凝蛋白II在纤维中取代Ser-to-Ala的杆状体将作为对照，对头部atp酶活性没有影响。我们在蛋白质化学章节LB中确定的是，野生型棒二聚体和三个磷酸化位点突变棒的热稳定性非常相似，使用222 nm的平衡圆二色性测量和差示扫描量热计（VP-DSC），慢扫描速率为4.89 C /小时。沉降速度实验表明，在pH 7.5、0.6 M KCl条件下制备的棒材均为二聚体，椭球轴比约为45。-肌球蛋白II棒，铰链突变体，磷酸化位点突变体，盘绕线圈，微丝，棘阿米巴，热稳定性，沉降

Acanthamoeba myosin II rod is a long alpha-helical dimeric coiled-coil with a flexible hinge containing a helix-breaking proline. Previously, the thermal stability of the complete wild-type rod domain of myosin II (residues 849-1509), a point mutant in which the hinge proline was replaced with alanine (P398A), and a deletion mutant with the whole hinge region deleted (residues 384-408) in 0.6 and 2.2 M KCl, pH 7.5, was determined. Analytical ultracentrifugation was used to characterize the size and shape (frictional ratio) of myosin II wild-type and hinge mutant rods. Sedimentation properties of minifilaments formed by dialysis of the wild-type and point and deletion mutants vs. low ionic strength buffer with 5 mM Mg (II) at pH 7.5 also have been determined. In addition, the flexibility of myosin II rod minifilaments as a function of Mg(II) concentration has been measured by electric birefringence; specifically, the Mg(II)-dependent flexible-to-stiff transitions of native myosin II and the wild-type rod minifilaments were found by Rau to be very similar. In the present studies, mutations of the phosphorylation sites in the C-terminal tail of the myosin II rod (with an amino terminal 694 residue flag) have substituted 3 Asp for 3 Ser [S1489D, S1494D, S1499D] (78,615 MW), 3 Ala for 3 Ser [S1489A, S1494A, S1499A] (78,483 MW), 1 Asp for Ser [S1489D] (78,559 MW); wild-type rod has 78,531 MW. The wild-type and mutant rods will be used by B. Kunnummal (LCB) to make cofilaments with native myosin II to test if the actin-activated ATPase activity of myosin II heads is inhibited by copolymerized rods containing the Ser-to-Asp substitutions (made to mimic phosphorylation at these sites). If so, the Ser-to-Ala substituted rods in cofilaments with myosin II will act as controls by showing no effect on the head ATPase activity. What we have determined in the Section on Protein Chemistry, LB, is that the thermal stabilities of the wild-type rod dimer and the three phosphorylation- site mutant rods are very similar, using equilibrium circular dichroism measurements at 222 nm and a differential scanning calorimeter (VP-DSC) at a slow scan rate of 4.89 C per hour. Sedimentation velocity experiments demonstrated that all rod preparations in 0.6 M KCl at pH 7.5 are dimeric with ellipsoidal axial ratios of about 45. - myosin II rod, hinge mutants, phosphorylation site mutants, coiled-coil, minifilaments, Acanthamoeba, thermal stability, sedimentation