MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2006; 44: 294–301 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1771 On the use of pseudocontact shifts in the structure determination of metalloproteins † Malene Ringkjøbing Jensen, 1 D. Flemming Hansen, 1 Umit Ayna, 2 Robert Dagil, 1 Mathias A. S. Hass, 1 Hans E. M. Christensen 3 and Jens J. Led 1* 1 Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark 2 Department of Chemistry, Bilkent University, 06800 Ankara, Turkey 3 Department of Chemistry, The Technical University of Denmark, Building 207, DK-2800 Kgs. Lyngby, Denmark Received 3 October 2005; Revised 13 November 2005; Accepted 15 November 2005 The utility of pseudocontact shifts in the structure refinement of metalloproteins has been evaluated using a native, paramagnetic Cu 2+ metalloprotein, plastocyanin from Anabaena variabilis (A.v.), as a model protein. First, the possibility of detecting signals of nuclei spatially close to the paramagnetic metal ion is investigated using the WEFT pulse sequence in combination with the conventional TOCSY and 1 H– 15 N HSQC sequences. Second, the importance of the electrical charge of the metal ion for the determination of correct pseudocontact shifts from the obtained chemical shifts is evaluated. Thus, using both the Cu + plastocyanin and Cd 2+ -substituted plastocyanin as the diamagnetic references, it is found that the Cd 2+ -substituted protein with the same electrical charge of the metal ion as the paramagnetic Cu 2+ plastocyanin provides the most appropriate diamagnetic reference signals. Third, it is found that reliable pseudocontact shifts cannot be obtained from the chemical shifts of the 15 N nuclei in plastocyanin, most likely because these shifts are highly dependent on even minor differences in the structure of the paramagnetic and diamagnetic proteins. Finally, the quality of the obtained 1 H pseudocontact shifts, as well as the possibility of improving the accuracy of the obtained structure, is demonstrated by incorporating the shifts as restraints in a refinement of the solution structure of A.v. plastocyanin. It is found that incorporation of the pseudocontact shifts enhances the precision of the structure in regions with only few NOE restraints and improves the accuracy of the overall structure. Copyright  2006 John Wiley & Sons, Ltd. KEYWORDS: NMR; 1 H; 13 C; 15 N; paramagnetic metalloprotein; blue copper protein; plastocyanin; pseudocontact shift; WEFT; SERF INTRODUCTION Paramagnetic metal ions are potential tools in the structure determination of proteins. In paramagnetic metalloproteins, the dipolar interactions between the unpaired electrons of the metal ion and the nuclei of the protein contain † Dedicated to Professor David M. Grant of the University of Utah on the occasion of his 75th birthday, in recognition of the outstanding contributions he has made to the methodology of nuclear magnetic resonance and its application to a wide range of chemical topics over a long period of time. L Correspondence to: Jens J. Led, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark. E-mail: led@kiku.dk Contract/grant sponsor: Danish Natural Science Research Council; Contract/grant numbers: 9400351, 9801801, 51-00211, 21-01-0545, and 21-04-0519. Contract/grant sponsor: Carlsbergfondet; Contract/grant number: 1624/40. Contract/grant sponsor: Novo Nordisk Fonden; Contract/grant number: 2003-11-28. Contract/grant sponsor: Villum Kann Rasmussen Fonden; Contract/grant number: 8.12.2003. valuable long-range structure information. In principle, these interactions can be detected by NMR spectroscopy through enhanced nuclear relaxation rates and changes in chemical shifts, i.e. the pseudocontact shifts. However, spin diffusion may complicate the use of para- magnetic relaxation enhancements in structure refinements of proteins (Hansen DF, Led JJ, unpublished results). Fur- thermore, fast exchange with solvent water may obscure the paramagnetic relaxation enhancement of amide protons. 1–3 In contrast, the pseudocontact shifts remain unaffected by these phenomena. Also, the pseudocontact shifts are experi- mentally more accessible, and can be determined accurately if an appropriate diamagnetic reference can be obtained. Recently, long-range pseudocontact shift restraints have been used as a supplement to the conventional nuclear Over- hauser enhancements (NOEs) and dihedral angle restraints 4 in structure refinements of metalloproteins, and proteins and nucleic acids with artificially incorporated paramagnetic metal ions. 5–11 Thus, the pseudocontact shifts have proved valuable in the refinement of structures where a sufficient number of NOEs are difficult to obtain. Also, pseudocontact shifts have been used to determine the relative orientation Copyright  2006 John Wiley & Sons, Ltd.