Browsing by Subject "binding"

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Open Access
Calcium Dependence of Fibrin Nanomechanics: The gamma 1 Calcium Mediates the Unfolding of Fibrinogen Induced by Force Applied to the "A-a" Bond
(2010) Akhremitchev, Boris B
The interactions between the constituent monomers of fibrin, the polymerized protein network that provides the structural stability of blood clots, ale frequently under stress because of the dynamic nature of blood flow Herein, the calcium dependence of the structural unfolding linked to the forced dissociation of the "A-a" knob-hole bond between fibrin monomers is reported The presence of calcium was shown to influence the incidence of the last event in the unfolding pattern characteristic of "A-a" rupture This effect, attributed to the function of the gamma 1 calcium-binding site, was found to be reversible and specific Our results indicate that binding of calcium at the gamma 1 site has no effect on the strength of the knob-hole bond prior to unfolding of the hole-containing gamma module Rather, calcium bound at the gamma 1 site makes the structure of the hole mole resilient to such forced unfolding, leading to survival of the "A-a" knob-hole bond during large: extensions of the fibrinogen molecule but at the cost of rupture of the bond at lower forces
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.