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

The de novo-designed metalloprotein alpha 3DIV 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.

Automated summary

The de novo-designed metalloprotein alpha 3DIV 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. It is found that any two of the three cysteines in the protein bind to mercury in the HgS2 site.