A MINIMAL MOTOR DOMAIN FROM CHICKEN SKELETAL-MUSCLE MYOSIN
A MINIMAL MOTOR DOMAIN FROM CHICKEN SKELETAL-MUSCLE MYOSIN
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DOI：
10.1074/jbc.270.25.15348
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发表时间：
1995-06-23
影响因子：
4.8
通讯作者：
LOWEY, S
中科院分区：
文献类型：
--
作者：
WALLER, GS;OUYANG, G;LOWEY, S
肌球蛋白头部（S1）由一个包含肌动蛋白和核苷酸结合位点的宽球状区域和一个α-螺旋延伸区域组成，该区域通过存在两类轻链而稳定。必需轻链邻接球状结构域，而调节轻链位于肌球蛋白的头-杆连接附近。通过温和变性剂去除必需轻链，使基础重链暴露于胰凝乳蛋白酶的蛋白水解。切割的片段，或“马达结构域”（MD），在SDS-聚丙烯酰胺凝胶电泳上作为单一条带迁移，其迁移率略高于木瓜蛋白酶或胰凝乳蛋白酶制备的S1。用MD修饰的肌动蛋白丝的三维图像分析揭示了类似于S1的结构，但是短了与不存在轻链结合结构域一致的量。肌动蛋白激活的MgATP酶活性在最大流速（V-max）和最大跨膜速率（K-m）上与S1相似。但是，在运动性测定中，MD移动肌动蛋白丝的能力相对于S1大大降低。我们的结论是，球状的，活性位点区域的肌球蛋白头是一个稳定的，独立折叠的结构域与内在的运动活性，但ATP水解和运动之间的耦合效率显着下降，轻链结合区被截断。
The myosin head (S1) consists of a wide, globular region that contains the actin- and nucleotide-binding sites and an alpha-helical, extended region that is stabilized by the presence of two classes of Light chains. The essential light chain abuts the globular domain, whereas the regulatory light chain lies near the head-rod junction of myosin. Removal of the essential Light chain by a mild denaturant exposes the underlying heavy chain to proteolysis by chymotrypsin. The cleaved fragment, or ''motor domain'' (MD), migrates as a single band on SDS-polyacrylamide gel electrophoresis, with a slightly greater mobility than S1 prepared by papain or chymotrypsin. Three-dimensional image analysis of actin filaments decorated with MD reveals a structure similar to S1, but shorter by an amount consistent with the absence of a Light chain-binding domain. The actin-activated MgATPase activity of MD is similar to that of S1 in V-max and K-m. But the ability of MD to move actin filaments in a motility assay is considerably reduced relative to S1. We conclude that the globular, active site region of the myosin head is a stable, independently folded domain with intrinsic motor activity, but the coupling efficiency between ATP hydrolysis and movement declines markedly as the light chain binding region is truncated.