Enhancing Ligand Epitope Mapping in Biomolecular Recognition by Variable Temperature Saturation Transfer Difference...

Saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy is a powerful and robust technique for the characterization of receptor-ligand interactions of medium to weak affinity in solution, providing binding epitope maps that reveal structural details of these interactions at atomic level. This knowledge is essential for understanding the structural basis of biomolecular recognition processes of biological relevance and plays a key role in drug discovery. Temperature is a critical factor influencing the sensitivity of STD NMR spectroscopy, as it directly affects the kinetics of complex formation, thereby modulating the observed STD effects. In this study, we systematically investigated the impact of temperature on STD NMR-derived binding epitope maps. Our findings highlight that, whenever protein stability permits, temperature should be considered an essential experimental parameter to be optimized for each system to obtain binding epitope maps with well-discriminated protein-ligand contact information. For the example cases studied herein, we demonstrate that temperature optimization allows to better assess the contribution of each individual contact to protein-ligand binding (enhanced ligand epitope mapping discrimination).