Abstract
Electrostatic interaction is a major driving force for several cases of protein binding to ligand, such as growth factor to cell surface glycosaminoglycan and DNA-binding protein to DNA. The free energy difference and 1H-15N HSQC perturbation of protein upon ligand titration are two critical physical quantities frequently employed for evaluating the binding contribution of a particular residue. However, based on the results shown in the previous studies, less correlation was found between the binding free energies and perturbed HSQC chemical shifts for those residues involved in charge-charge interactions. The reason might be due to that the changes observed in 1H-15N HSQC spectra, revealing the protein backbone resonances, could not represent the interactions occurring in the end of long side chain, especially for the Lys and Arg long side chain moieties. In order to clarify the discrepancy, we first utilize the NMR pulse sequence H2CN, providing information available for the realistic events arising from amino acid side chains rather than protein backbone by using conventional HSQC titration experiments, to probe the charge-charge interactions through the non-exchangeable protons in lysine residues binding to the counterion ligands. In the present study, we choose hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2) as the protein models, which possess 70% and 30% heparin binding affinities contributed from electrostatic interactions, respectively. Our data indicate that in the H2CN titration experiments, most of chemical shift perturbations occurring in lysine/argine side chains show a good correlation to the free energy changes of single-Lys/Arg mutants for both HDGF and bFGF, suggesting that NMR H2CN provides a precise and quick evaluation of electrostatic interactions in heparin-binding proteins. 3