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. 2022 Jan 11;15(5):4251–4257. doi: 10.1007/s12274-021-4065-9

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy

Jingyuan Nie 1, Yibing Deng 1, Fang Tian 1, Shengchao Shi 1, Peng Zheng 1,✉
PMCID: PMC9077643  PMID: 35574260

Abstract

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.

graphic file with name 12274_2021_4065_Fig1_HTML.jpg

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.

Keywords: cation-π interaction, neutrophil gelatinase-associated lipocalin (NGAL), single-molecule force spectroscopy, atomic force microscopy (AFM)

Electronic Supplementary Material

12274_2021_4065_MOESM1_ESM.pdf (987.4KB, pdf)

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy

Acknowledgments

This work was funded by the Fundamental Research Funds for the Central Universities (No. 14380259), Natural Science Foundation of Jiangsu Province (No. BK20200058), the National Natural Science Foundation of China (Nos. 21771103 and 21977047), and computational resources from computing facilities of the High-Performance Computing Center (HPCC) of Nanjing University.

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Supplementary Materials

12274_2021_4065_MOESM1_ESM.pdf (987.4KB, pdf)

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy

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