Residue-level elucidation of the ligand-induced protein binding on phenyl-argarose microspheres by NMR hydrogen/deuterium exchange technique

Protein-ligand interactions on liquid-solid interfaces governed the design of functional biomaterials. However, accurate residue details of ligand induced protein binding and unfolding on an interface were still unknown by the current ensemble of protein structure characterizations. Here, a hydrogen/deuterium (H/D) approach coupled with analysis of NMR TOCSY spectra and the solvent accessible surface area (SASA) was designed to enable residue level understanding of lysozyme adsorbed at a phenyl-ligand modified surface. Results showed that the binding sites and unfolding of lysozyme molecules on phenyl-agarose microspheres demonstrated significant ligand-density dependence and protein-coverage dependence. Either increasing ligand density or decreasing adsorption coverage would lead to more binding sites and unfolding of the protein molecules. With the multipoint adsorption strengthening, the protein molecule changed from lying end-on to side-on. Finally, Molecular Dock simulation was utilized to evaluate the NMR determined binding sites based on energy ranking of the binding. It confirmed that this NMR approach represents a reliable route to in silico abundant residue-level structural information during protein interaction with biomaterials.