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

我们通过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。该配合物的旋转半径为49 A，表观分子量为165 kD。基于两分子的刚体旋转和平移，寻找与整个散射曲线吻合较好的actin和S1原子结构模型。该模型的唯一性通过不对称dna - i -actin和S1的复合体来检验。S1的尖端与肌动蛋白亚结构域1结合，正如先前的肌凝蛋白分子的电子显微镜所预期的那样，该分子的头部装饰有单体g -肌动蛋白。(98) 123, 8)。在ADP的作用下，Rg降低了3a，但分子量没有变化。最佳拟合模型是将两个分子在S1长轴上相互扭曲近90度，同时保留它们的主要结合位点。二聚体或二聚体肌动蛋白与S1的复合体现在被检查，结晶正在进行中。计算机拟合了肌肉纤维中自旋标记肌球蛋白轻链的ESR谱，并结合了两个等摩尔高斯分布。松弛肌纤维的光谱符合不对称分布，并分解成两个宽角分布，表明一对肌球蛋白轻链结构域（颈部区域）在肌球蛋白丝主干上以不同方向松散固定。现在分析活动肌纤维的频谱。利用定点诱变技术对微管蛋白结合区进行自旋标记。从自旋标记ESR估计的侧链迁移率受到微管蛋白结合的限制，固定依赖于微管上激酶的占用，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.