Two Histidines in an α-Helix: A Rigid Co²⁺-Binding Motif for PCS Measurements by NMR Spectroscopy

Pseudocontact shifts (PCS) generated by paramagnetic metal ions present valuable long-range information in the study of protein structural biology by nuclear magnetic resonance (NMR) spectroscopy. Faithful interpretation of PCSs, however, requires complete immobilization of the metal ion relative to the protein, which is difficult to achieve with synthetic metal tags. We show that two histidine residues in sequential turns of an α-helix provide a binding site for a Co²⁺ ion, which positions the metal ion in a uniquely well-defined and predictable location. Exchange between the bound and free cobalt is slow on the timescale defined by chemical shifts, but the NMR resonance assignments are nonetheless readily transferred from the diamagnetic to the paramagnetic NMR spectrum by an I_zS_z-exchange experiment. The double-histidine-Co²⁺ motif offers a straightforward, inexpensive, and convenient way of generating precision PCSs in proteins.