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Updated: Aug 12, 2025
Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
Published on: February 28, 2019
Quantification of carboxylate-bridged di-zinc site stability in protein due ferri by single-molecule force
Zhiyi Wang1, Mengdie Wang1, Zhongxin Zhao1
1State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, People's Republic of China.
Abstract:
Carboxylate-bridged diiron proteins belong to a protein family involved in different physiological processes. These proteins share the conservative EXXH motif, which provides the carboxylate bridge and is critical for metal binding. Here, we choose de novo-designed single-chain due ferri protein (DFsc), a four-helical protein with two EXXH motifs as a model protein, to study the stability of the carboxylate-bridged di-metal binding site. The mechanical and kinetic properties of the di-Zn site in DFsc were obtained by atomic force microscopy-based single-molecule force spectroscopy. Zn-DFsc showed a considerable rupture force of ~200 pN, while the apo-protein is mechanically labile. In addition, multiple rupture pathways were observed with different probabilities, indicating the importance of the EXXH-based carboxylate-bridged metal site. These results demonstrate carboxylate-bridged di-metal site is mechanically stable and improve our understanding of this important type of metalloprotein.
