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

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.