Quantification of carboxylate-bridged di-zinc site stability in protein due ferri by single-molecule force spectroscopy

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 similar to 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.

Automated summary

Carboxylate-bridged diiron proteins are a family of proteins involved in various physiological processes. These proteins have a conserved EXXH motif that forms the carboxylate bridge and is crucial for metal binding. In this study, we used a de novo-designed single-chain di-ferri protein (DFsc) with two EXXH motifs as a model protein to investigate the stability of the carboxylate-bridged di-metal binding site. By using atomic force microscopy-based single-molecule force spectroscopy, we obtained the mechanical and kinetic properties of the di-Zn site in DFsc. The results demonstrate the mechanical stability of the carboxylate-bridged di-metal site and enhance our understanding of this important type of metalloprotein.