STRUCTURAL STUDIES OF MOTOR PROTEINS

描述（摘自申请）：我们的项目是确定运动蛋白家族运动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，这些小分子诱导驱动蛋白分子改变其结构，而不是已经通过x射线衍射成像的ADP-kinesin和ADP-NCD复合物。我们的实验将集中在三个运动蛋白上：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运动马达和人类运动蛋白，在神经元中传递囊泡的正向运动马达，将用ATP类似物与微管蛋白肽片段或微管蛋白二聚体结晶。Kcm1是一种马达蛋白同源物，它是一种分解附着在着丝点上的微管的机器，是一种拆卸机器。这很有趣，因为它结合并稳定了微管蛋白二聚体。这些x射线结构对了解运动机制很重要。在复合物中加入微管蛋白或肽片段是必要的，因为运动蛋白用于运动的构象变化是由其与ATP和微管蛋白的相互作用产生的。KCM1是结构研究的新目标蛋白，是突变因子蛋白组的一员。KCM1与激酶蛋白的不同之处在于，它在多肽序列中有一个中央运动结构域，在C端有一个短卷曲的螺旋茎，在n端有一个与染色体的着丝点结合的球状结构域。我们的策略是获得NCD、激酶和KCM1的纯结构域，并在大肠杆菌中表达。纯化后的蛋白将用于检测atp酶活性和与微管蛋白二聚体的结合。微管蛋白将从禽红细胞中制备，因为这种样品是相对等基因和均匀的。我们将纯化这些配合物并使其结晶，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.