Excellent Fe(II) Binding Tag in Protein Paramagnetic NMR Spectroscopy

Most first series of transition metal ions have one or more unpaired electrons and show great variations in the paramagnetic property. The magnetic anisotropy of some transition metal ions, including Co(II), as well as lanthanide ions [Ln(III)], has been well examined in proteins by NMR. In contrast, few examples of Fe(II) complexes reporting the magnetic anisotropy were analyzed in proteins, except for the ones containing a heme motif or iron-sulfur clusters. Here, we showed that [2,2':6',2″-terpyridine]-6,6″-dicarboxylic acid (TDA) is an excellent iron-binding ligand. It forms a stable iron complex in aqueous solution and demonstrates distinct iron-binding properties. TDA forms a 1:1 stable complex with both Fe(II) and Fe(III), but Fe(II) presents a high-spin state in the complex. The TDA moiety can be site-specifically attached to a protein, and its protein conjugate generates sizable pseudocontact shifts (PCSs) in complex with Fe(II), which are larger than those of commonly used metal binding tags. In contrast, the protein-TDA-Fe(III) complex produces negligible paramagnetic relaxation enhancement (PRE) effects in the protein signals, indicating a low-spin state of Fe(III) in the protein-TDA complex. The high stability of the protein-TDA-Fe(II) complex allows one to measure accurate PCSs in cell lysate even in the presence of other transition metal ions and an excess of GSH.