ABSTRACT
Physiological processes rely on initial recognition events between cellular components and other molecules or modalities. Biomolecules can have multiple sites or mode of interaction with other molecular entities, so that a resolution of the individual binding events in terms of spatial localization as well as association and dissociation kinetics is required for a meaningful description. Here we describe a trichromatic fluorescent binding- and displacement assay for simultaneous monitoring of three individual binding sites in the important transporter and binding protein human serum albumin. Independent investigations of binding events by X-ray crystallography and time-resolved dynamics measurements (switchSENSE technology) confirm the validity of the assay, the localization of binding sites and furthermore reveal conformational changes associated with ligand binding. The described assay system allows for the detailed characterization of albumin-binding drugs and is therefore well-suited for prediction of drug-drug and drug-food interactions. Moreover, conformational changes, usually associated with binding events, can also be analyzed.
Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Boron Compounds/chemistry , Ibuprofen/chemistry , Lauric Acids/chemistry , Serum Albumin, Human/chemistry , Warfarin/chemistry , 4-Chloro-7-nitrobenzofurazan/chemistry , Binding Sites , Crystallography, X-Ray , Fluorescence , Humans , Molecular Dynamics Simulation , Molecular StructureABSTRACT
A single high-affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C12 fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site-specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA-binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow-sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X-ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug-protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.
