X-ray Study of Actin Monomer-Myosin Complex and ESR Angular Measurement of Alkaline Light Chains

我们用MBS和DHT制备了一种不可聚合的G-肌动蛋白，并测量了1：1肌动蛋白-肌球蛋白S1复合物的X射线溶液散射。复合物的回转半径（Rg）为49 A，表观分子量为165 kD.根据两分子的刚体转动和平移，寻找了与整个散射曲线拟合较好的由肌动蛋白和S1原子结构组成的模型.该模型的唯一性通过不对称DNaseI-肌动蛋白和S1的复合物来检查。S1的尖端与肌动蛋白亚结构域1结合，正如从先前的肌球蛋白分子的电子显微镜所预期的，该肌球蛋白分子的头部用单体G-肌动蛋白修饰（J.Struct.Biol.（'98）123，8）。在ADP存在下，Rg下降了3A，分子量没有变化.通过将两个分子围绕S1的长轴彼此扭转近90度并保留其主要结合位点来获得最佳拟合模型。二聚体或三聚体肌动蛋白和S1的复合物现在正在研究中，结晶正在进行中。用两个等摩尔高斯分布的组合对肌纤维中自旋标记肌球蛋白轻链的ESR谱进行计算机拟合。放松的肌纤维的光谱与不对称分布拟合，并分解成两个宽的角分布，这表明一对肌球蛋白轻链结构域（颈部区域）在肌球蛋白丝骨架上的不同方向上松散地固定。现在分析来自活动肌纤维的光谱。驱动蛋白的微管蛋白结合区域使用定点突变进行自旋标记。由自旋标记ESR估计的侧链移动性受到微管蛋白结合的限制，固定化依赖于驱动蛋白在微管上的占据，这表明结构变化沿着微管传播。

We made a nonpolymerizable G-actin by MBS and DHT and measured X-ray solution scattering from a 1 : 1 acto-myosin S1 complex. The radius of gyration (Rg) of the complex was 49 A and apparent molecular weight was 165 kD.The model consisted of atomic structures of actin and S1 which fits the whole scattering curve closely was searched on the basis of rigid-body rotation and translation of two molecules. The uniqueness of the model was checked by a complex of an asymmetric DNaseI-actin and S1. A tip of S1 binds to actin subdomain 1 as expected from the previous electron microscopy of a myosin molecule whose heads are decorated with a monomeric G-actin (J.Struct.Biol. ('98)123, 8). In the presence of ADP, Rg decreased by 3 A with no change in molecular weight. The best fit model was obtained by twisting the two molecules each other through nearly 90 degrees about a long axis of S1 with retaining their main binding site. The complex of dimeric or timeric actin and S1 is now examined, and crystallization is in progress. The computer-fitting of ESR spectra from spin-labeled myosin light-chain in muscle fibres was done by a combination of two equimolar Gaussian distributions. The spectrum of relaxed muscle fibres was fitted with an asymmetric distribution and resolved into two broad angular distributions, suggesting that a pair of the myosin light-chain domains (neck regions) appear fixed loosely at different orientations on the myosin filament backbone. The spectrum from active muscle fibres is now analyzed. The tubulin-binding region of kinesin was spin-labeled using site-directed mutagenesis. A side-chain mobility estimated from spin-label ESR was restricted by tubulin binding, The immobilization depends on the occupancy of kinesin on a microtubule, suggesting a propagation of structural change along microtubule.