On the use of pseudocontact shifts in the structure determination of metalloproteins.
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.
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Malene Ringkjøbing
MR
Jensen
D Flemming
DF
Hansen
Umit
U
Ayna
Robert
R
Dagil
Mathias A S
MA
Hass
Hans E M
HE
Christensen
Jens J
JJ
Led
D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010970:Plastocyanin / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D010970:Plastocyanin