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
