Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface‐ and solution‐based biophysical methods

Abstract

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface-and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

“…Here we characterize the binding of a flexible diphosphorylated ITAM peptide and a rigid ITAM-derived ligand to the Syk tSH2 domain by thermodynamic and kinetic analysis by SPR, as well as by studying effects on protein dynamics with ESI-MS. From previous work by us and by others it appeared that such an SPR approach can give reliable thermodynamic data. [37,38] The affinity of Syk tSH2 for g-ITAM in solution was found to be identical to that for immobilized g-ITAM (1) at the SPR sensor surface. This has been observed previously for single SH2 domains, [38] and indicates that the affinity is not influenced by additional effects of the sensor chip.…”

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

“…The validity of the SPR approach has been rigorously established. One study in this regard [45] showed that the kinetic, equilibrium, and thermodynamic constants determined for the interaction of tethered carbonic anhydrase II with arylsolfonamide compounds closely matched those obtained using two solution-based biophysical methods, isothermal titration calorimetry and stopped flow fluorescence.…”

Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases

“…Lysinemethylated T was probably immobilized in its free N-terminus using standard amine coupling chemistry. (1) Transition state analysis was carried out using the Eyring equations, which assume an active state in equilibrium with reactants (A + B ↔AB* ↔ AB) whose concentration determines the velocity of the reaction [21]. This should be considered simply as a convenient semi-empirical model in the current context.…”

“…While the formation of the BTCI/-CT complex is exothermic and favored by an entropy increase (Hvh = -7.5  0.9 kcal.mol -1 and Svh= 18  3 cal.mol -1 .K -1 ), the BTCI/T interaction is endothermic and therefore driven by a large entropy increase (Hvh= 8  2 kcal.mol -1 and Svh= 70  6 cal.mol -1 .K -1 ). Eyring equations were used to dissect the reaction pathways by introducing a theoretical transient state (Figure 2A and 2B) [21]. Theoretical reaction pathways were built based on transition state theory and they are depicted in Figure 3. …”