Comparison of ncd and kinesin motor domains by circular dichroism spectroscopy.
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.
Shimizu
T
T
National Institute of Bioscience and Human-Technology, Ibaraqi.
Morii
H
H
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004398:Dyneins / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism; D008868:Microtubule Proteins / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D029721:Drosophila Proteins; D008868:Microtubule Proteins; C066696:ncd protein, Drosophila; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D004398:Dyneins; D016547:Kinesins