Shimizu T, Morii H
National Institute of Bioscience and Human-Technology, Ibaraqi.
J Biochem. 1996 Dec;120(6):1176-81. doi: 10.1093/oxfordjournals.jbchem.a021538.
ncd is a microtubule motor protein from Drosophila, having a 40 kDa domain homologous to the kinesin motor domain. In the present study, we investigated the circular dichroism (CD) spectra of the ncd motor domain in comparison with those of the kinesin motor domain. Although the two are about 40% identical in amino acid sequence, and recent X-ray crystallographic studies [Sablin, Kull, Cooke, Vale, and Fletterick (1996) Nature 380, 555-559; Kull, Sablin, Lau, Fletterick, and Vale (1996) Nature 380, 550-555] indicate that their core structures are nearly identical, the far UV CD spectra of ncd and kinesin motor domains, both being monomeric, were considerably different from each other, suggesting a significant difference in the secondary, especially loop structures. The motor domain of ncd, like that of kinesin, contains tightly associating ADP even after purification. We removed ADP from the ncd motor domain by gel filtration in the presence of EDTA and high salt. The resultant protein, however, was likely to be in an inactive state, since it bound ATP slowly. The far UV CD spectrum of the ncd motor domain devoid of ADP was nearly identical to that of the ncd motor domain with bound ADP. This indicated that the removal of ADP did not affect the backbone structure in the presence of high salt. On the other hand, the near UV CD spectrum of the ADP-free ncd motor domain differed from that of the ncd motor domain. ADP complex, one possibility being that the local conformation was changed upon removal of bound ADP. The near UV CD spectra of kinesin motor domain also showed a difference between the ADP-bound form and the nucleotide-free form, although the difference was much smaller.
Ncd是一种来自果蝇的微管运动蛋白，具有一个与驱动蛋白运动结构域同源的40 kDa结构域。在本研究中，我们研究了Ncd运动结构域的圆二色性（CD）光谱，并与驱动蛋白运动结构域的光谱进行了比较。尽管两者在氨基酸序列上约有40%的同一性，且最近的X射线晶体学研究[萨布林、库尔、库克、瓦尔和弗莱泰里克（1996年）《自然》380卷，555 - 559页；库尔、萨布林、刘、弗莱泰里克和瓦尔（1996年）《自然》380卷，550 - 555页]表明它们的核心结构几乎相同，但Ncd和驱动蛋白运动结构域的远紫外CD光谱（两者均为单体形式）却有很大差异，这表明在二级结构，尤其是环结构上存在显著差异。与驱动蛋白一样，Ncd的运动结构域即使在纯化后也紧密结合着ADP。我们在EDTA和高盐存在的情况下，通过凝胶过滤从Ncd运动结构域中去除了ADP。然而，所得蛋白质可能处于无活性状态，因为它结合ATP的速度很慢。不含ADP的Ncd运动结构域的远紫外CD光谱与结合了ADP的Ncd运动结构域的光谱几乎相同。这表明在高盐存在的情况下，去除ADP不会影响主链结构。另一方面，不含ADP的Ncd运动结构域的近紫外CD光谱与Ncd运动结构域 - ADP复合物的光谱不同，一种可能性是在去除结合的ADP后局部构象发生了变化。驱动蛋白运动结构域的近紫外CD光谱在结合ADP形式和无核苷酸形式之间也显示出差异，尽管差异要小得多。
