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An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a -barrel metalloprotein

¹ Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202-Elche (Alicante), Spain
² Center of Magnetic Resonance (CERM), University of Florence, 50019 Sesto Fiorentino (Florence), Italy
³ Departamento de Química Inorgánica, Universitat de València, 46100 Burjassot (Valencia), Spain

(RECEIVED January 5, 2005; FINAL REVISION March 29, 2005; ACCEPTED April 8, 2005)

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D₂O/H₂O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide protons. In contrast, the last loop (including a short -helix) and the last -strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.

Keywords: rusticyanin; blue copper protein; NMR; protein unfolding; protein dynamics

Abbreviations: Az, azurin • BCP, blue copper protein • GdmCl, guanidinium chloride • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser effect • Rc, rusticyanin

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051337505.

Reprint requests to: Antonio Donaire, Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Edificio Torregaitán, Avda. de la Universidad s/n, 03202-Elche (Alicante), Spain; e-mail: adonaire{at}umh.es; fax: +34-96-6658758.

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