Inverse PI by NMR: Analysis of Ligand 1H-Chemical Shifts in the Protein-Bound State

The study of protein-ligand interactions via protein-based NMR generally relies on the detection of chemical-shift changes induced by ligand binding. However, the chemical shift of the ligand when bound to the protein is rarely discussed, since it is not readily detectable. In this work we use protein deuteration in combination with [1H-1H]-NOESY NMR to extract 1H chemical shift values of the ligand in the bound state. The chemical shift perturbations (CSPs) experienced by the proton ligand resonances (free vs bound) are an extremely rich source of information on protein-ligand complexes. Besides allowing for the detection of intermolecular CH-π interactions and elucidation of the protein-bound ligand conformation, the CSP information can be used to analyse (de)solvation effects in a site-specific manner. In conjunction with crystal structure information, it is possible to distinguish protons whose desolvation penalty is compensated for upon protein-binding, from those that are not. Combined with the previously reported PI by NMR technique for the protein-based detection of intermolecular CH-π interactions, this method represents another important step towards the ultimate goal of Interaction-Based Drug Discovery.