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Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles
Author(s) -
BaergaOrtiz Abel,
Bergqvist Simon,
Mandell Jeffrey G.,
Komives Elizabeth A.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03120604
Subject(s) - isothermal titration calorimetry , chemistry , allosteric regulation , kinetics , enthalpy , thrombin , binding site , plasma protein binding , biophysics , thermodynamics , biochemistry , enzyme , physics , platelet , quantum mechanics , biology , immunology
Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein–protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K D s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the Δ G bind was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the Δ G bind from electrostatic steering was assessed from the dependence of the k a on ionic strength. Release of solvent H 2 O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein–protein binding. The mAb binding is enthalpy driven and has a slow k d . TM binding appears to be entropy driven and has a fast k a . The favorable entropy of the thrombin–TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.