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

The specific interaction between Ni-nitrilotriacetic acid and the six-histidine tag may be one of the most important coordination bonds utilized in biological research because of its wide application for recombinant protein purification. The complex stability is critical for target protein binding. Thus, measurement of the mechanical stability of the system was attempted soon after the invention of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) two decades ago. Moreover, the competing ligand imidazole and protons are the two critical factors for target protein elution. However, the mechanochemistry between the system and the imidazole/proton has not been determined. Here, an AFM-SMFS system using strain-promoted alkyne–azide cycloaddition and Cu-free click chemistry was used to characterize the system. Consequently, the destabilizing effect of the imidazole and proton on the interaction was revealed quantitatively, leading to a 3-fold increase in the bond dissociation rate

Single-Molecule Force Spectroscopy Reveals the Dynamic HgS Coordination Site in the <i>De Novo</i>-Designed Metalloprotein α₃DIV

The de novo-designed metalloprotein α3DIV binds to mercury via three cysteine residues under dynamic conditions. An unusual trigonal three-coordinate HgS3 site is formed in the protein in basic solution, whereas a linear two-coordinate HgS2 site is formed in acidic solution. Furthermore, it is unknown whether the two coordinated cysteines in the HgS2 site are fixed or not, which may lead to more dynamics. However, the signal for HgS2 sites with different cysteines may be similar or may be averaged and indistinguishable. To circumvent this problem, we adopt a single-molecule approach to study one mercury site at a time. Using atomic force microscopy-based single-molecule force spectroscopy, the protein is unfolded, and the HgS site is ruptured. The results confirm the formation of HgS3 and HgS2 sites at different pH values. Moreover, it is found that any two of the three cysteines in the protein bind to mercury in the HgS2 site

Training results of tri-classification of BCCD images under different epoch and batch size.

Training results of tri-classification of BCCD images under different epoch and batch size.</p

ROC curve.

(a) MobileNetV2. (b) ResNet. (c) SE-ResNeXt.</p

WBC-AMNet visualization analysis of attention to different feature maps.

WBC-AMNet visualization analysis of attention to different feature maps.</p

ROC curve.

(a) MobileNetV2. (b) ResNet. (c) SE-ResNeXt.</p

Statistical results of nine classic CNN models.

Statistical results of nine classic CNN models.</p

Training results when epoch = 20 and batch size = 32.

Training results when epoch = 20 and batch size = 32.</p

Training results of different WBC subtypes.

Training results of different WBC subtypes.</p

Comparison of CNN structure between WBC-AMNet and other models.

Comparison of CNN structure between WBC-AMNet and other models.</p