Large Heat Capacity Change in a Protein−Monovalent Cation Interaction

Abstract

Current views about protein-ligand interactions state that electrostatic forces drive the binding of charged species and that burial of hydrophobic and polar surfaces controls the heat capacity change associated with the reaction. For the interaction of a protein with a monovalent cation the electrostatic components are expected to be significant due to the ionic nature of the ligand, whereas the heat capacity change is expected to be small due to the size of the surface area involved in the recognition event. The physiologically important interaction of Na+ with thrombin was studied over the temperature range from 5 to 45 degrees C and the ionic strength range from 50 to 800 mM. These measurements reveal an unanticipated result that bears quite generally on studies of molecular recognition and protein folding. Binding of Na+ to thrombin is characterized by a modest dependence on ionic strength but a large and negative heat capacity change of -1.1 +/- 0.1 kcal mol-1 K-1. The small electrostatic coupling can be explained in terms of a minimal perturbation of the ionic atmosphere of the protein upon Na+ binding. The large heat capacity change, however, is difficult to reconcile with current views on the origin of this effect from surface area changes or large folding transitions coupled to binding. It is proposed that this change is linked to burial of a large cluster of water molecules in the Na+ binding pocket upon Na+ binding. Due to their reduced mobility and highly ordered structure, water molecules sequestered in the interior of a protein must have a lower heat capacity compared to those on the surface of a protein or in the bulk solvent. Hence, a binding or folding event where water molecules are buried may result in significant heat capacity changes independent of changes in exposed hydrophobic surface or coupled conformational transitions.

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

“…Consistent with this interpretation, tight-binding exosite I ligands such as TM are still capable of converting these variants into a catalytically competent state. This disordered-to-ordered structural transition involving approximately 50 residues explains the high negative heat capacity associated with thrombin ligation (24), and is consistent with previous studies using hydrogen/deuterium exchange to monitor the effect of ligation on thrombin structure (25,26). In contrast, NMR studies on the analogous unregulated serine protease trypsin revealed no conformational or dynamic differences upon binding to activesite inhibitors (27).…”

“…4G). However, the most dramatic effect of exosite I occupancy is the stabilization of the C-terminal β-barrel (partial or full ordering of five of the six strands), including the N terminus (18)(19)(20)(21)(22)(23)(24), the 170s loop and helix (163-178), the 180s loop (182-189), and the N-and C-terminal stocks of the γ-loop (138-146 and 154-157). However, only part of the Na þ -binding loop is ordered by exosite I binding, including residues 213-215 and 226-228.…”

Rapid Kinetics of Na+ Binding to Thrombin

“…The value of K app can also be derived from equilibrium titrations of intrinsic fluorescence (35, 39 -41, 46, 47) or linkage studies (38,46,48). The value of K A , on the other hand, can only be derived from rapid kinetic studies.…”

Molecular Dissection of Na+ Binding to Thrombin

“…Under physiologic conditions of [Na ϩ ] (140 mM), pH and temperature, the K d for Na ϩ binding to thrombin is 110 mM (6,30,31), which implies that nearly half of the thrombin molecules generated in vivo from prothrombin are in the Na ϩ -free slow form. This basic property of thrombin was discovered in 1992 (6), but since then the Protein Data Bank has accumulated Ͼ150 structures of the enzyme that, with only one exception, have been solved in the presence of Na ϩ and portray thrombin in the fast form.…”