Quantification of the Effects of Ionic Strength, Viscosity, and Hydrophobicity on Protein–Ligand Binding Affinity

Abstract

In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (K d) is essential. Estimation of the binding affinity in this way is routinely performed in "favorable" conditions for macromolecules. Crucial data for ligand-protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the K d of three previously characterized protein-ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity.

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans

“…Binding was completely reconstituted upon the addition of 25 mM calcium chloride (CaCl 2 ) (Fig. 1E), though binding decreased in the presence of higher concentrations of CaCl 2 , possibly because of the changes in ionic strength affecting protein conformation and/or binding (32). EGTA treatment did not affect the binding of anti-mouse IgG to mouse IgG Fc (data not shown).…”

Comparison of zonal elution and nonlinear chromatography in determination of the interaction between seven drugs and immobilised β2-adrenoceptor

“…Other methods include crystallography [7], spectrofluorometry [8], affinity capillary electrophoresis [9], molecular docking [10] and nuclear magnetic resonance [11]. In addition to these methods, several new technologies such as surface plasmon resonance [12], isothermal titration calorimetry [13], biosensors [14] and quartz crystal microbalances [15] are used for exploring the thermodynamic and kinetic characteristics of drug-protein interactions.…”

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones

“…These would result in a high background, which would dilute the fluorescence signal, thus masking the effect of ligand binding to cyclin A. The octapeptide HAKRRLIF [9], the pentapeptide RRLIF[34] and the N-capped peptide SCCP5921 (this study), were the three ligands sampled for titration, representing high, low and putatively intermediate affinity compounds. HAKRRLIF represents an optimized version of the cyclin binding motif found in the endogenous CDK2 inhibitor, p21Waf, whereas RRLIF represents a truncated version of this sequence retaining respectable activity at a lower molecular weight and is therefore considered as a good compromise between potency and size [15, 35].…”