How to Distinguish Conformational Selection and Induced Fit Based on Chemical Relaxation Rates

Protein binding often involves conformational changes. Important questions are whether a conformational change occurs prior to a binding event (‘conformational selection’) or after a binding event (‘induced fit’), and how conformational transition rates can be obtained from experiments. In this article, we present general results for the chemical relaxation rates of conformational-selection and induced-fit binding processes that hold for all concentrations of proteins and ligands and, thus, go beyond the standard pseudo-first-order approximation of large ligand concentration. These results allow to distinguish conformational-selection from induced-fit processes—also in cases in which such a distinction is not possible under pseudo-first-order conditions—and to extract conformational transition rates of proteins from chemical relaxation data.Author Summary: The function of proteins is affected by their conformational dynamics, i.e. by transitions between lower-energy ground-state conformations and higher-energy excited-state conformations of the proteins. Advanced NMR and single-molecule experiments indicate that higher-energy conformations in the unbound state of proteins can be similar to ground-state conformations in the bound state, and vice versa. These experiments illustrate that the conformational change of a protein during binding may occur before a binding event, rather than being induced by this binding event. However, determining the temporal order of conformational transitions and binding events typically requires additional information from chemical relaxation experiments that probe the relaxation kinetics of a mixture of proteins and ligands into binding equilibrium. These chemical relaxation experiments are usually performed and analysed at ligand concentrations that are much larger than the protein concentrations. At such high ligand concentrations, the temporal order of conformational transitions and binding events can only be inferred in special cases. In this article, we present general equations that describe the dominant chemical relaxation kinetics for all protein and ligand concentrations. Our general equations allow to clearly infer from relaxation data whether a conformational transition occurs prior to a binding event, or after the binding event.