Calprotectin’s Protein Structure Shields Ni–N(His) Bonds from Competing Agents

The Ni–N­(His) coordination bond, formed between the nickel ion and histidine residues, is essential for recombinant protein purification, especially in Ni-NTA-based systems for selectively binding polyhistidine-tagged (Histag) proteins. While previous studies have explored its bond strength in a synthetic Ni-NTA-Histag system, the influence of the surrounding protein structure remains less understood. In this study, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to quantify the Ni–N­(His) bond strength in calprotectin, a biologically relevant protein system. Our results demonstrate that the Ni–N­(His) bond in protein exhibits a rupture force of ∼56 pN. Notably, kinetic analysis revealed a significantly lower off-rate compared to the synthetic system, suggesting that the protein environment plays a crucial role in stabilizing the bond. Moreover, we found that the bond is less susceptible to displacement by competing agents, such as imidazole, and experiences only a modest decrease in stability under acidic conditions, compared to the dramatic weakening seen in a synthetic system. These findings highlight the role of protein structure in protecting the mechanical and kinetic stability of the Ni–N­(His) bond, offering insights into understanding the metal–ligand interactions in proteins in general.