Exploring weak ligand-protein interactions by relaxometry of long-lived spin order

Relaxation times of nuclear spins often serve as a valuable source of information on the dynamics of various biochemical processes. Measuring relaxation as a function of the external magnetic field turned out to be extremely useful for the studies of weak ligand-protein interactions. We demonstrate that observing the relaxation of the long-lived spin order instead of longitudinal magnetization extends the capability of this approach. We studied the field-dependent relaxation of the longitudinal magnetization and the singlet order (SO) of methylene protons in alanine-glycine dipeptide and citrate in the presence of human serum albumin (HSA). As a result, SO relaxation proved to be more sensitive to ligand-protein interaction, providing higher relaxation contrast for various HSA concentrations. To assess the parameters of the binding process in more details, we utilized a simple analytical relaxation model to fit the experimental field dependences for both SO and T 1 relaxation. We also tested the validity of our approach in the experiments with trimethylsilylpropanoic acid (TSP) used as a competitor in ligand binding with HSA. Measuring relaxation as a function of the external magnetic field detects weak ligand-protein interactions. Employing long-lived spin order instead of longitudinal magnetization extends this method's capabilities.