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 alpha-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 H-1-N-15 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 Angstrom, respectively, and decrease to 0.39 +/- 0.05 and 0.90 +/- 0.06 Angstrom 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 Angstrom. Backbone NH R-1, R-2, 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 Angstrom resolution. All differences are related to local changes in the amino acidic composition.
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