Analyzing multi-step ligand binding reactions for oligomeric proteins by NMR: Theoretical and computational...

Solution NMR spectroscopy is widely used to investigate the thermodynamics and kinetics of the binding of ligands to their biological receptors, as it provides detailed, atomistic information, potentially leading to microscopic affinities for each binding event, and, to the development of allosteric pathways describing how the binding at one site affects distal sites in the molecule. Importantly, weak interactions that are often invisible to other biophysical methods can also be probed. Methodological advancements in NMR have enabled the investigation of high molecular weight, homo-oligomeric complexes that bind multiple ligand molecules, with increasing numbers of studies of the structural dynamics and binding properties of these systems. It therefore becomes of interest to consider how binding and kinetics parameters can be extracted from experiments on these more complicated molecules. Here we present the theoretical framework for analyzing binding reactions of homo-oligomeric complexes by NMR, taking into account all of the chemical species in solution and their corresponding NMR observables. A number of simulations are presented to illustrate the utility of the derived expressions.