title: Paramagnetism-based refinement strategy for the solution structure of human alpha-parvalbumin.
abstract: 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 Ca(2+) ion in the EF site with the paramagnetic Dy(3+) ion. A simple (1)H-(15)N HSQC spectrum allowed the NH assignments based on the properties of Dy(3+). 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 A, respectively, and decrease to 0.39 +/- 0.05 and 0.90 +/- 0.06 A 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 A. 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 A resolution. All differences are related to local changes in the amino acidic composition.
authors.affiliation: Magnetic Resonance Centre and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy.
authors.forename: Irfan
authors.initials: I
authors.lastname: Baig
authors.forename: Ivano
authors.initials: I
authors.lastname: Bertini
authors.forename: Cristina
authors.initials: C
authors.lastname: Del Bianco
authors.forename: Yogesh Kumar
authors.initials: YK
authors.lastname: Gupta
authors.forename: Yong-Min
authors.initials: YM
authors.lastname: Lee
authors.forename: Claudio
authors.initials: C
authors.lastname: Luchinat
authors.forename: Alessandro
authors.initials: A
authors.lastname: Quattrone
mesh_terms: D000577:Amides / Q000737:chemistry; D000818:Animals; D016880:Anisotropy; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D018360:Crystallography, X-Ray; D004419:Dysprosium / Q000737:chemistry; D020832:EF Hand Motifs; D006801:Humans; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010320:Parvalbumins / Q000737:chemistry*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry; D011522:Protons; D051381:Rats; D012996:Solutions
chemical_list: D000577:Amides; D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010320:Parvalbumins; D020033:Protein Isoforms; D011522:Protons; D012996:Solutions; D004419:Dysprosium