Spectroscopic studies of the ncd motor domain center dot 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 N-6-methylADP (M eADP) which has a UV absorption spectrum different from that of ADP. The re sultant 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 ind icates that the bound nucleotide contributes to the near-UV CD spectra to a considerable extent although ADP or MeADP free in solution gives a spectru m 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 contri buted to the spectral changes of ADP. A candidate chromophore is Tyr(442) w hich is stacked with the adenine moiety at a distance of 0.43 nm. On the ot her hand, we detected an intensity decrease of tryptophanyl fluorescence up on binding of a nucleotide to the nucleotide-free ncd motor domain, while a t the same time tyrosyl fluorescence increased. The fluorescence changes, a s well as the UV absorption change described above, gave similar rates upon addition of a nucleotide to the nucleotide-free ncd motor domain. Therefor e, they are likely to originate from the same conformational change of the protein.