The iron−amino acid interactions Fe−O(Glu/Asp), Fe−N(His), and Fe− S(Cys) are the three major iron−ligand bonds in proteins. To compare their properties in proteins, we used atomic force microscopy (AFM)-based single-molecule force spectroscopy to investigate a superoxide reductase (Fe(III)-SOR) with all three types of bonds forming an Fe(His) 4 CysGlu center. We first found that Apo-SOR without bound iron showed multiple unfolding pathways only from the β-barrel core. Then, using Holo-SOR with a ferric ion, we found that a single Fe−O(Glu) bond can tightly connect the flexible N-terminal fragment to the β-barrel and stabilize the whole protein, showing a complete protein unfolding scenario, while the single Fe−N(His) bond was weak and unable to provide such a stabilization. Moreover, when multiple Fe−N bonds are present, a similar stabilization effect can be achieved. Our results showed that the iron−ligand bond modulates protein structure and stability in different modes at the single-bond level.

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α3D is a de-novo designed three-helix bundle protein. Like most naturally occurring helical proteins, it is mechanically labile with an unfolding force of <15pN, revealed by atomic force microscopy-based single-molecule...

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Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene. It plays an essential role in many biological systems as a vital driving force for protein folding, stability, and receptor-ligand interaction/recognition. To date, the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional (3D) protein structures and corresponding computational calculations. However, their experimental verification and quantification remain sparse at the molecular level, mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins. Here, we use atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of protein neutrophil gelatinase-associated lipocalin (also known as NGAL, siderocalin, lipocalin 2) that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols. Based on a site-specific cysteine engineering and anchoring method, we first characterized the stability and unfolding pathways of apo-NGAL. Then, the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized. AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites, F122 and F133. Here, NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect. Thus, our work demonstrates high-precision detection of the weak cation-π interaction in NGAL. Electronic Supplementary Material Supplementary material (additional SDS-PAGE, UV-vis, protein sequences, and more experimental methods) is available in the online version of this article at 10.1007/s12274-021-4065-9.