Biochemical and Biological Properties of Myosins

我们以前发现，嵌合体中的心肌病（CM）环的网骨藻肌球蛋白II被替换的CM环的平滑肌或心肌肌球蛋白II的生化损伤在体外和功能受损在体内。此外，Ala 400到瓦尔的单点突变在生物化学和生物学上几乎完全失活，而心脏嵌合体中的回复突变Val 400 Ala几乎完全恢复了该失活嵌合体的活性，尽管事实上它在CM环中的11个残基中的7个中仍然不同。对acto-S1催化的ATP酶循环的瞬时动力学分析表明，在所有嵌合体中，受影响的步骤是从acto-S1、ADP、Pi中间体释放P1。在400位存在瓦尔也大大降低了S1对F-肌动蛋白的亲和力。这些结果表明，CM-环的构象会影响运动域的整体构象，从而影响肌动蛋白和肌球蛋白之间的整体互补性。 阿米巴肌球蛋白IC的尾部含有一个碱性区（BR）、一个甘氨酸-脯氨酸-丙氨酸富集区（GPA 1）、一个SH 3区和一个GPA 2区。冷冻电子显微镜的先前结果表明，在具有GPA 1和GPA 2的BR与BR相互作用后，尾部折叠回自身。N15-和N15/C13-标记的全长尾和单独表达的N-末端和C-末端半尾的NMR数据的初步分析与该解释一致，与单独的半尾相比，全长尾中骨架酰胺原子的化学位移和强度存在显著差异。 阿米巴肌球蛋白II是由磷酸化的三个丝氨酸残基的C-末端非螺旋尾片段。我们正试图了解这种情况发生的机制，因为调节丝氨酸残基与它们通过约90 nm的卷曲螺旋调节的ATP酶位点分离。用天冬氨酸或谷氨酸替换丝氨酸对尾部构建体的生物物理性质没有影响。重链和两条轻链的cDNA在Sf 9细胞中共表达，具有良好的完全活性、可调节的肌球蛋白II产量。表达的酶是完全活跃时，监管丝氨酸被替换为无论是谷氨酸或谷氨酸，表明无论是天冬氨酸或谷氨酸模拟磷酸丝氨酸在这种情况下。

We had found previously that chimeras in which the cardiomyopathy (CM) loop of Dictyostelium myosin II was replaced by the CM loop of either smooth muscle or cardiac muscle myosin II were biochemically impaired in vitro and functionally impaired in vivo. Also, the single point mutation of Ala400 to Val was almost completely inactive both biochemically and biologically while the reverse mutation, Val400Ala, in the cardiac chimera restored almost complete activity to this inactive chimera despite the fact that it still differed in 7 of the 11 residues in the CM-loop. Transient kinetic analysis of the ATPase cycle catalyzed by acto-S1 showed that the affected step in all of the chimeras was the release of P1 from the acto-S1.ADP.Pi intermediate. The presence of Val at position 400 also greatly reduced the affinity of S1 for F-actin in the absence of nucleotide. These results indicate that the conformation of the CM-loop affects the overall conformation of the motor domain affecting the overall complementarity between actin and myosin. The tail of Acanthamoeba myosin IC contain a basic region (BR) followed by a glycine-proline-alainine rich region (GPA1), and SH3 region and a GPA2 region. Previous results from cryoelectron microscopy indicated that the tail was folded back on itself after the BR with GPA1 and GPA2 interacting with BR. Preliminary analysis of NMR data of N15-, and N15/C13-labeled full length tail and the separately expressed N-terminal and C-terminal halves of the tail are consistent with this interpretations there are significant differences in chemical shifts and intensities of backbone amide atoms in the full-length tail compared to the separate halves. Acanthamoeba myosin II is regulated by phosphorylation of three serines residues in the C-terminal non-helical tailpiece. We are trying to understand the mechanism by which this occurs given that the regulatory serine residues are separated from the ATPase site they regulated by about 90 nm of coiled-coil helix. Replacing the serines with either aspartate or glutamate had no effect on the biophysical properties of tail constructs. cDNAs for the heavy chain and two light chains were co-expressed in Sf9 cells with good yield of fully active, regulatable myosin II. Expressed enzyme was fully active when the regulatory serines were replaced by either aspartates or glutamates, indicating that neither Asp nor Glu mimics phosphoSer in this situation.