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 microscopybased 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.

Lead is a toxic metal harmful to human beings because of its long history and wide use in human society. The interaction of lead with proteins is one of the most common ways it exerts toxicity. Due to its thiophilic property, lead targets thiol-rich proteins with high affinity and forms stable Pb−S bonds, which may replace the originally bound metal ion and incapacitate the protein/enzyme. Thus, the knowledge of the Pb−S bonds in proteins is important. To study Pb−S bonds, we chose a de novo designed protein α 3 DIV, able to bind Pb(II) via its three cysteines, as the model protein.Using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), we proved the formation of a triangular pyramidal PbS 3 site in α 3 DIV by detecting specific Pb−S bond rupture signals, including the previously undetected Pb− S(Cys67) bond. Moreover, the pH-dependent weakening of Pb−S bond strength was revealed and quantified, leading to the dissociation of the PbS 3 site at pH 4.5.