STRUCTURAL STUDIES OF MOTOR PROTEINS

描述（摘自申请）：我们的项目是确定驱动蛋白家族马达蛋白的原子水平结构。我们将使用单晶X射线晶体学来确定驱动蛋白与蛋白质、肽或小分子的蛋白质复合物的结构，这些蛋白质、肽或小分子诱导驱动蛋白分子从已经通过X射线衍射成像的ADP-驱动蛋白和ADP-NCD复合物改变其结构。我们的实验将集中在三个驱动蛋白：两个是分子马达，一个是微管拆卸机。NCD，进行减数分裂所需的反向行进马达和人驱动蛋白，在神经元中递送囊泡的正向行进马达，将用具有微管蛋白的肽片段或微管蛋白二聚体的ATP类似物结晶。 Kcm 1是一种马达蛋白同源物，它是一种在微管附着于动粒上时分解微管的机器，也是一种拆卸机器。这是有趣的，因为它结合并稳定微管蛋白二聚体。这些X射线结构对于理解运动机制非常重要。在复合物中包括微管蛋白或肽片段是必要的，因为驱动蛋白用于运动的构象变化是通过其与ATP和微管蛋白的相互作用产生的。KCM 1是结构研究的新蛋白质，是突变因子蛋白质组的成员。KCM 1与驱动蛋白的不同之处在于在多肽序列中具有一个中央马达结构域，在C末端具有一个短卷曲螺旋茎，N末端具有一个结合染色体动粒的球状结构域。我们的策略是获得NCD、驱动蛋白和KCM 1的纯结构域，并在E.杆菌将测定纯化的蛋白质的ATP酶活性和与微管蛋白二聚体的结合。将从禽类红细胞制备微管蛋白，因为该样品相对等基因且均质。我们将纯化复合物并使其结晶用于X射线衍射研究。

Description (taken from the applications): Our project is to determine atomic level structures for kinesin family motor proteins. We will use single crystal X-ray crystallography to determine structures of protein complexes of kinesins with proteins, peptides or small molecules that induce the kinesin molecule to change its structure from the ADP-kinesin and ADP-NCD complexes already imaged by X-ray diffraction. Our experiments will focus on three kinesins: two are molecular motors and one is a microtuble disassembly machine. NCD, the reverse traveling motor required to carry out meiosis and human kinesin, the forward traveling motor that delivers vesicles in neurons are to be crystallized with ATP analogs with peptide fragments of tubulin or with tubulin dimers. Kcm1, a motor protein homolog that is a machine for breaking up microtubules while attached to kinetochores is the disassembly machine. It is interesting because it binds to and stabilizes tubulin dimers. These X-ray structures are important for understanding of the mechanisms of motility. Including tubulin or peptide fragments in the complexes is essential because4 the conformational changes that kinesins use for motility are generated by its interactions with ATP and tubulin. KCM1, the new protein targeted for structural studies, is a member of the group of proteins that are catastrophe factors. KCM1 is distinct from kinesins in having a central motor domain in the polypeptide sequence with a short coiled coil stalk at the C terminus and an N-terminal globular domain that binds to kinetochores of chromosomes. Our strategy is to obtain pure domains of NCD, kinesin and KCM1 and to express these in E. coli. The purified proteins will be assayed for ATPase activity and for binding to tubulin dimers. Tubulin will be prepared from avian erythrocytes as this sample is relatively isogenic and homogeneous. We will purify the complexes and crystallize them for X-ray diffraction studies.