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Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles

¹ Department of Chemistry, Cambridge University, Cambridge, UK
² Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0378, USA
³ Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA

Reprint requests to: Elizabeth A. Komives, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA; e-mail: ekomives{at}ucsd.edu; fax: (858) 534-6174.

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_Ds, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the G_bind was measured by isothermal titration calorimetry and van’t Hoff analysis. The contribution to the G_bind from electrostatic steering was assessed from the dependence of the k_a on ionic strength. Release of solvent H₂O 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.

Keywords: Surface plasmon resonance; calorimetry; amide H/²H exchange; MALDI-TOF mass spectrometry; hydration; entropy; enthalpy

Abbreviations: TM, thrombomodulin • EGF, epidermal growth factor • MALDI-TOF, matrix assisted laser desorption ionization time-of-flight • MS, mass spectrometry • H/²H, hydrogen/deuterium

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