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 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 AFM-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. This article is protected by copyright. All rights reserved.