ACANTHAMOEBA MYOSIN II ROD AND MUTANTS: PHYSICAL PROPERTIES

棘阿米巴肌球蛋白II棒是一个长的α-螺旋二聚体卷曲线圈，带有一个柔性铰链，含有一个螺旋断裂的脯氨酸。以前，肌球蛋白II的完整野生型杆状结构域(残基849-1509)、铰链脯氨酸被丙氨酸取代的点突变体(P398A)和整个铰链区缺失的缺失突变体(残基384-408)在0.6和2.2M KCl0.6和2.2MKCl、pH 7.5中的热稳定性被测定。用分析超速离心法对肌球蛋白II野生型和铰链突变棒的大小和形状(摩擦率)进行表征。还测定了由野生型、点突变体和缺失突变体与含5 mM镁(II)的低离子强度缓冲液在pH 7.5时透析形成的微丝的沉降性能。此外，通过电双折射测量了肌球蛋白II杆状微丝的柔韧性随镁离子浓度的变化；具体地说，RAU发现天然肌球蛋白II和野生型杆状微丝的依赖于镁(II)的柔韧性到刚性的转变非常相似。在目前的研究中，肌球蛋白II棒C末端的磷酸化位点的突变(带有一个氨基酸末端694个残基标志)已经用3 Asp取代了3 Ser[S1489D，S1494D，S1499D](78,615 mW)，3 Ala替代了3 Ser[S1489A，S1494A，S1499A](78,483 mW)，1 Asp替代了Ser[S1489D](78,559 mW)；野生型棒有78,531 mW。野生型和突变的杆状物将被B.kunnummal(LCB)用来与天然肌球蛋白II制造共丝，以测试肌球蛋白II头部的肌动蛋白激活的ATPase活性是否被含有Ser-to-Asp取代(模仿这些位点的磷酸化)的共聚杆所抑制。如果是这样的话，肌球蛋白II在共丝中的Ser-to-Ala取代棒将作为对照，对头部ATPase活性没有影响。我们在LB的蛋白质化学部分确定的是，野生型杆状二聚体和三个磷酸化位点突变的杆状分子的热稳定性非常相似，使用222 nm处的平衡圆二色谱测量和差示扫描量热仪(VP-DSC)在每小时4.89℃的慢扫描速度下进行测量。沉降速度实验表明，在0.6M KCl中、pH 7.5的溶液中制备的棒材均为二聚体，椭球轴向比约为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、沉降性

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