title: Effect of urea on protein-ligand association.
abstract: We combine experimental and theoretical approaches to investigate the influence of a cosolvent on a ligand-protein association event. We apply fluorescence measurements to determining the affinity of the inhibitor tri-N-acetylglucosamine [(GlcNAc)3] for lysozyme at urea concentrations ranging from 0 to 8M. Notwithstanding that, at room temperature and neutral pH, lysozyme retains its native conformation up to the solubility limit of urea, the affinity of (GlcNAc)3for the protein steadily decreases as the concentration of urea increases. We analyze the urea dependence of the binding free energy within the framework of a simplified statistical thermodynamics-based model that accounts for the excluded volume effect and direct solute-solvent interactions. The analysis reveals that the detrimental action of urea on the inhibitor-lysozyme binding originates from competition between the free energy contributions of the excluded volume effect and direct solute-solvent interactions. The free energy contribution of direct urea-solute interactions narrowly overcomes the excluded volume contribution thereby resulting in urea weakening the protein-ligand association. More broadly, the successful application of the simple model employed in this work points to the possibility of its use in quantifying the stabilizing/destabilizing action of individual cosolvents on biochemical folding and binding reactions.
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada.
authors.forename: Lora
authors.initials: L
authors.lastname: Stepanian
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada.
authors.forename: Ikbae
authors.initials: I
authors.lastname: Son
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. Electronic address: chalikan@phm.utoronto.ca.
authors.forename: Tigran V
authors.initials: TV
authors.lastname: Chalikian
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D012997:Solvents; D014508:Urea; D009113:Muramidase; D000117:Acetylglucosamine
keywords: Cosolvents; Lysozyme; Protein-ligand binding; Thermodynamics; Urea