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ACS Med Chem Lett. 2014 Jul 9;5(8):931-6. doi: 10.1021/ml500204e. eCollection 2014 Aug 14.

Quantification of the effects of ionic strength, viscosity, and hydrophobicity on protein-ligand binding affinity.

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Veterinary School, University of Thessaly , Trikalon 224, Karditsa 43100, Greece ; Institute for Research and Technology-Thessaly (I.RE.TE.TH.), The Centre for Research & Technology Hellas (CE.R.TH.) , Dimitriados 95 & Paulou Mela, Volos 383 33, Greece.
Veterinary School, University of Thessaly , Trikalon 224, Karditsa 43100, Greece.
Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina , Columbia, South Carolina 29202, United States.


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.


Affinity; dissociation constant; fluorescence spectroscopy; ion strength; solvent; viscosity

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