Paramagnetism-Based Refinement Strategy for the Solution Structure of Human α-Parvalbumin

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.