In the frame of a research aimed at the detailed structural characterization of human calcium-binding proteins of the EF-hand family, the solution structure of human α-parvalbumin has been solved by NMR and refined with the help of substitution of the Ca2+ ion in the EF site with the paramagnetic Dy3+ ion. A simple 1H- 15N HSQC spectrum allowed the NH assignments based on the properties of Dy3+. This allowed us to exploit pseudocontact shifts and residual dipolar couplings for solution structure refinement. The backbone and heavy atom RMSD are 0.55 ± 0.08 and 1.02 ± 0.08 Å, respectively, and decrease to 0.39 ± 0.05 and 0.90 ± 0.06 Å upon refinement with paramagnetism-based restraints. The RMSD for the metal itself in the EF site in the refined structure is 0.26 ± 0.12 Å. Backbone NH R1, R2, and NOE measured at two temperatures show the protein to be relatively rigid. The NH orientations are well determined by the paramagnetism-based restraints. This allows us to detect small but significant local structural differences with the orthologue protein from rat, whose X-ray structure is available at 2.0 Å resolution. All differences are related to local changes in the amino acidic composition.
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