Single-Molecule Force Spectroscopy Reveals the Dynamic HgS Coordination Site in the De Novo -Designed Metalloprotein α 3 DIV.

The de novo -designed metalloprotein α 3 DIV binds to mercury via three cysteine residues under dynamic conditions. An unusual trigonal three-coordinate HgS 3 site is formed in the protein in basic solution, whereas a linear two-coordinate HgS 2 site is formed in acidic solution. Furthermore, it is unknown whether the two coordinated cysteines in the HgS 2 site are fixed or not, which may lead to more dynamics. However, the signal for HgS 2 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 HgS 3 and HgS 2 sites at different pH values. Moreover, it is found that any two of the three cysteines in the protein bind to mercury in the HgS 2 site.