Conformational kinetics reveals affinities of protein conformational states.
Abstract
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.
Type
Journal articleSubject
allosterybinding
change
conformational
coupled
Allosteric Site
Bacterial Proteins
Binding Sites
Calorimetry
Drug Design
Kinetics
Ligands
Macromolecular Substances
Magnetic Resonance Spectroscopy
Models, Molecular
Protein Binding
Protein Conformation
Protein Folding
Ribonuclease P
Thermodynamics
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https://hdl.handle.net/10161/10594Published Version (Please cite this version)
10.1073/pnas.1502084112Publication Info
Daniels, Kyle G; Suo, Yang; & Oas, Terrence G (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 https://hdl.handle.net/10161/10594.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
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 pro

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