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

Jingyuan Nie; Yibing Deng; Fang Tian; Shengchao Shi; Peng Zheng

doi:10.1007/s12274-021-4065-9

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

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

Authors

Jingyuan Nie¹, Yibing Deng¹, Fang Tian¹, Shengchao Shi¹, Peng Zheng¹

Affiliation

¹ State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, 163 Xianlin Road, Nanjing, 210023 China.

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.

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