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Updated: Sep 20, 2025
OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
Published on: February 5, 2020
Single-Molecule Force Spectroscopy Reveals the Dynamic HgS Coordination Site in the De Novo-Designed Metalloprotein
Ziyi Wang1, Zhongxing Zhao1, Guoqiang Li1
1State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, P.R. China.
Abstract:
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.
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