Abstract
We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone 15N T 1/T 2 data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T 1/T 2 ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1•DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.
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Kikuchi, J., Iwahara, J., Kigawa, T. et al. Solution structure determination of the two DNA-binding domains in the Shizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints. J Biomol NMR 22, 333–347 (2002). https://doi.org/10.1023/A:1014977808170
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DOI: https://doi.org/10.1023/A:1014977808170