Chapter 12. Long-lived States-based Determination of Ligand Affinity for a Target Protein

Abstract

The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. The use of long-lived states (LLS) for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with KD in the millimolar range, where most other biophysical techniques fail. Furthermore, the combination of LLS for screening with 1H dissolution-DNP to enhance the sensitivity is an interesting application for measuring LLS signals of weak ligands while using very low concentrations of ligands and proteins. Dramatic differences have been observed between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, LLS involving pairs of 19F nuclei have been explored to study binding phenomena. Fluorinated ligands can be used as spy molecules in competition experiments, which allowed the ranking of affinities and the estimation of the dissociation constants of arbitrary ligands that do not contain any fluorine.