Browsing by Subject "coupled"

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Open Access
Conformational kinetics reveals affinities of protein conformational states.
(Proc Natl Acad Sci U S A, 2015-07-28) Daniels, Kyle G; Suo, Yang; Oas, Terrence G
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.
Open Access
Kinetics of Coupled Binding and Conformational Change in Proteins and RNA
(2015) Daniels, Kyle Gabriel
Ligand binding can modulate function of proteins and nucleic acids by changing both the populations of functionally distinct conformational states and the timescales on which they interconvert. For this reason, both thermodynamic and kinetic details of coupling can be important to proper function. How tightly does ligand bind to the different conformational states? What effect does ligand binding have on the conformational equilibrium and conformational kinetics? On what timescales and in what order do binding and conformational change occur? Using a combination of stopped-flow kinetics, isothermal titration calorimetry, and x-ray crystallography, we determine the mechanisms of coupled binding and conformational change in protein (Bacillus subtilis RNase P protein) and RNA (DP17 biosensor) systems.
The results demonstrate that rigorous kinetic analysis can be used to estimate the equilibrium and rate constants for conformational changes, as well as the affinities of ligands for different conformational states. A single ligand can bind to different conformational states of the same protein or nucleic acid with affinities that differ by orders of magnitude. This binding shifts the conformational equilibrium towards the higher affinity state through a combination of increasing rate constants for the forward conformational change and decreasing rate constants for the reverse conformational change. Using a flux-based analysis of the mechanisms we show that molecular recognition is kinetically partitioned between a number of pathways that differ by the order in which binding and conformational change occur. The absolute and relative flux through these pathways varies with ligand concentration, the affinities of the ligand for the various conformational states, and the ability of ligand to accelerate the conformational change. Together, the results give insights into how biological function depends on the kinetic and thermodynamic details of coupled binding and conformational change.