Spectroscopic studies of the ncd motor domain.ADP complex: CD spectrum of ADP induced by binding to the motor domain of ncd.
Previously, we reported that the nucleotide-free ncd motor domain exhibited a near-UV CD spectrum different from that of the ordinary ncd motor domain.ADP complex [Shimizu and Morii, (1996) J. Biochem. 120, 1176-1181]. In the present study, we exchanged the bound nucleotide ADP with N6-methylADP (MeADP) which has a UV absorption spectrum different from that of ADP. The resultant ncd motor domain. MeADP complex gave a near-UV CD spectrum different from that of the ordinary ncd motor domain with bound ADP. This result indicates that the bound nucleotide contributes to the near-UV CD spectra to a considerable extent although ADP or MeADP free in solution gives a spectrum with negligible peaks and troughs. In addition, the absorption intensity of ADP or MeADP at the peak wavelengths decreased to a considerable extent upon binding to the nucleotide-free ncd motor domain. It is suggested that interaction between adenine moiety and chromophore(s) of the protein contributed to the spectral changes of ADP. A candidate chromophore is Tyr442 which is stacked with the adenine moiety at a distance of 0.43 nm. On the other hand, we detected an intensity decrease of tryptophanyl fluorescence upon binding of a nucleotide to the nucleotide-free ncd motor domain, while at the same time tyrosyl fluorescence increased. The fluorescence changes, as well as the UV absorption change described above, gave similar rates upon addition of a nucleotide to the nucleotide-free ncd motor domain. Therefore, they are likely to originate from the same conformational change of the protein.
Shimizu
T
T
National Institute of Bioscience and Human-Technology, Ibaraqi, Japan.
Morii
H
H
D000244:Adenosine Diphosphate / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005456:Fluorescent Dyes / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C066696:ncd protein, Drosophila; D014443:Tyrosine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D003402:Creatine Kinase; D016547:Kinesins