State 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.
Protein Sci. 2023 Mar;32(3):e4583. doi: 10.1002/pro.4583.
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.
羧酸桥联双核铁蛋白属于参与不同生理过程的蛋白质家族。这些蛋白质共享保守的 EXXH 基序，该基序提供羧酸桥，对于金属结合至关重要。在这里，我们选择从头设计的单链亚铁蛋白 (DFsc)，一种具有两个 EXXH 基序的四螺旋蛋白作为模型蛋白，来研究羧酸桥联双核金属结合位点的稳定性。通过基于原子力显微镜的单分子力谱技术获得了 DFsc 中二锌位点的力学和动力学性质。Zn-DFsc 表现出相当大的断裂力 (~200 pN)，而脱辅基蛋白在力学上不稳定。此外，观察到具有不同概率的多种断裂途径，表明基于 EXXH 的羧酸桥联金属位点的重要性。这些结果表明羧酸桥联双核金属位点在力学上是稳定的，并提高了我们对这种重要类型的金属蛋白的理解。
