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

Abstract

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.