Lanthanide-induced pseudocontact shifts for solution structure refinementsof macromolecules in shells up to 40 angstrom from the metal ion

A number of pseudocontact shifts (PCS) in monolanthanide-substituted Calbin din D-9k (Ca2Cb henafter), a protein of 75 amino acids, were measured for C e(III), Yb(III), and Dy(III). The assignment of the shifts was obtained thr ough the conventional assignment procedures for the Ce(IH) derivative (CaCe Cb), since the line broadening is not severe, whereas in the case of Dy(III ) and Yb(III) the assignment was obtained by analyzing the temperature depe ndence of the H-1-N-15 HSQC shifts of the lanthanide derivatives and compar ing the results with the H-1-N-15 HSQC spectrum of Ca:Cb or CaCeCb. The NOE -based solution structures of Ca::Cb or CaCeCb were then refined with PCS. Since the three lanthanides span a wide range of magnetic anisotropies, the refinement was effective in shells from the metal of similar to 5-15 Angst rom for Ce(III), similar to 9-25 Angstrom for Yb(III), and similar to 13-40 Angstrom for Dy(III), as useful PCS were observed in these shells. The roo t-mean-square deviation of 30 conformers from the average for CaCeCb was 0. 74 and 1.10 Angstrom for the backbone and all heavy atoms, respectively, ob tained from 1539 NOEs, 39 (3)J values, and 6 T-1 values. With 589 pseudocon tact shifts for ce(III) (out of which 280 were larger than 0.1 ppm), 92 PCS for Yb(III), and 74 for Dy(III) the RMSD decreased to 0.54 and 0.95 A for Ce(III), 0.60 and 0.98 Angstrom,for Yb(III), and 0.66 and 1.04 Angstrom for Dy(mj for the backbone and all heavy atoms, respectively. While for Ce(III ) resolution improvements are mainly found for the metal binding site itsel f Yb(III) and Dy(III) can further constrain regions far away from the metal . These results show that constructing a lanthanide binding site may be a g eneral and convenient tool to "enlighten" shells at variable distances from the metal itself and may be used for various purposes including the invest igation of biomolecular complexes.