Conformational kinetics reveals affinities of protein conformational states

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 30; pp. 9352 - 9357
• Main Authors: Daniels, Kyle G, Yang Suo, Terrence G. Oas
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 28.07.2015; National Acad Sciences
• Subjects: Allosteric Site; allostery; Bacterial Proteins - chemistry; binding; binding capacity; Binding Sites; Biological Sciences; Calorimetry; change; conformational; coupled; Drug Design; drugs; Kinetics; Ligands; Macromolecular Substances; Magnetic Resonance Spectroscopy; Models, Molecular; nucleic acids; Protein Binding; Protein Conformation; Protein Folding; Proteins; Ribonuclease P - chemistry; Thermodynamics; binding; coupled; change; allostery; conformational
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1502084112

Despite 50 years of studies of coupled binding and conformational change reactions in proteins and nucleic acids, there has been no detailed analysis of the affinities of the multiple conformational states involved. The relationship between these affinities and the dynamics of conformational change has also been largely overlooked. We perform rigorous kinetic analysis of protein conformational change coupled to binding to concomitantly determine binding affinities of individual conformational states and rate constants for conformational changes. The results point to an inextricable link between affinities of the individual states and the change in conformational dynamics upon binding. The difference in affinities of conformational states is critical for driving conformational change and has implications in molecular recognition, allostery, and drug development. Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.