Biological function of proteins relies on conformational transitions and binding of specific ligands. Protein-ligand interactions are thermodynamically and kinetically coupled to conformational changes in protein structures as conceptualized by the models of pre-existing equilibria and induced fit. NMR spectroscopy is particularly sensitive to complex ligand-binding modes—NMR line-shape analysis can provide for thermodynamic and kinetic constants of ligand-binding equilibria with the site-specific resolution. However, broad use of line shape analysis is hampered by complexity of NMR line shapes in multi-state systems. To facilitate interpretation of such spectral patterns, I computationally explored systems where isomerization or dimerization of a protein (receptor) molecule is coupled to binding of a ligand. Through an extensive analysis of multiple exchange regimes for a family of three-state models, I identified signature features to guide an NMR experimentalist in recognizing specific interaction mechanisms. Results also show that distinct multistate models may produce very similar spectral patterns. I also discussed aggregation of a receptor as a possible source of spurious three-state line shapes and provided specific suggestions for complementary experiments that can ensure reliable mechanistic insight
