The binding ability of a protein with a metal binding tag towards Ni2+ was investigated by longitudinal paramagnetic NMR relaxation, and the possibility of obtaining long-range structure information from the paramagnetic relaxation was explored. A protein with a well-defined solution structure ( Escherichia coli thioredoxin) was used as the model system, and the peptide His-His-Pro (HHP) fused to the N-terminus of the protein was used as the metal binding tag. It was found that the tag forms a stable dimer complex with the paramagnetic Ni2+ ion, where each metal ion binds two HHP-tagged protein molecules. However, it was also found that additional sites in the protein compete with the HHP-tag for the binding of the metal ion. These binding sites were identified as the side chain carboxylate groups of the aspartic and glutamic acid residues. Yet, the carboxylate groups bind the Ni2+ ions considerably weaker than the HHP-tag, and only protons spatially close to the carboxylate sites are affected by the Ni2+ ions bound to these groups. As for the protons that are unaffected by the carboxylate-bound Ni2+ ions, it was found that the long-range distances derived from the paramagnetic relaxation enhancements are in good agreement with the solution structure of thioredoxin. Specifically, the obtained long-range paramagnetic distance constraints revealed that the dimer complex is asymmetric with different orientations of the two protein molecules relative to the Ni2+ ion.
