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

作　　者：Jingyuan Nie Yibing Deng Fang Tian Shengchao Shi Peng Zheng 

机构地区：[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

出　　处：《Nano Research》2022年第5期4251-4257,共7页纳米研究（英文版）

基　　金：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);computational resources from computing facilities of the High-Performance Computing Center(HPCC)of Nanjing University。

摘　　要：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.

关 键 词：cation-Tr interaction neutrophil gelatinase-associated lipocalin(NGAL) single-molecule force spectroscopy atomic force microscopy(AFM) 

分 类 号：O53[理学—等离子体物理]