Effect of the Competing Ligand on Ni-NTA/Histag Strength Revealed by Click Chemistry-Based Force Spectroscopy

The specific interaction between Ni-nitrilotriaceticacid and thesix-histidine tag may be one of the most important coordination bondsutilized in biological research because of its wide application forrecombinant protein purification. The complex stability is criticalfor target protein binding. Thus, measurement of the mechanical stabilityof the system was attempted soon after the invention of atomic forcemicroscopy-based single-molecule force spectroscopy (AFM-SMFS) two decades ago. Moreover, the competing ligand imidazole and protonsare the two critical factors for target protein elution. However,the mechanochemistry between the system and the imidazole/proton hasnot been determined. Here, an AFM-SMFS system using strain-promotedalkyne-azide cycloaddition and Cu-free click chemistry wasused to characterize the system. Consequently, the destabilizing effectof the imidazole and proton on the interaction was revealed quantitatively,leading to a 3-fold increase in the bond dissociation rate.

Automated summary

The specific interaction between Ni-nitrilotriaceticacid and the six-histidine tag is widely used in biological research for recombinant protein purification. The stability of this interaction is crucial for target protein binding, while the mechanochemistry between the system and the competing ligand imidazole/proton remains unclear. In this study, an AFM-SMFS system was used to characterize the system and reveal the destabilizing effect of imidazole and proton on the interaction, leading to a 3-fold increase in the bond dissociation rate.