Conformational kinetics reveals affinities of protein conformational states.

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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.





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Daniels, Kyle G, Yang Suo and Terrence G Oas (2015). Conformational kinetics reveals affinities of protein conformational states. Proc Natl Acad Sci U S A, 112(30). pp. 9352–9357. 10.1073/pnas.1502084112 Retrieved from

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Terrence Gilbert Oas

Professor of Biochemistry

Our laboratory is primarily interested in the mechanisms of protein folding. We use nuclear magnetic resonance (NMR) and other types of spectroscopy to study the solution structure, stability and folding reactions of small protein models. These include monomeric λ repressor, the B domain of protein A (BdpA) and various regulator of G-protein signalling (RGS) domains. Our biophysical studies are used to inform our investigations of the role of folding mechanism in the function of proteins in the cell. For example, a naturally occuring cancer-causing mutation in the RGS domain of axin appears to lower the thermodynamic stability of the domain. We are developing methods to compensate for such destabilizing mutations, thereby restoring normal function to the protein.We are also developing computational models of protein folding as a way to better understand the mechanisms and as a tool in the design of new experiments.

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