key: cord-0077825-lfv2nup8
authors: Nie, Jingyuan; Deng, Yibing; Tian, Fang; Shi, Shengchao; Zheng, Peng
title: Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy
date: 2022-01-11
journal: Nano Res
DOI: 10.1007/s12274-021-4065-9
sha: 46872372ac52020eeb3280da1468cb2db65ce4ea
doc_id: 77825
cord_uid: lfv2nup8

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. [Image: see text] 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.

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Mahadevi, A. S.; Sastry, G. N. Cation-π interaction: Its role and relevance in chemistry, biology, and material science. Chem. Rev. 2013, 113, 2100-2138. [4] Dougherty, D. A. The cation-π interaction. Acc. Chem [46]

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