title: ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.
abstract: The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit.
authors.affiliation: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Winiewska
authors.affiliation: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Bugajska
authors.affiliation: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Jarosław
authors.identifier: http://orcid.org/0000-0003-2684-1775
authors.initials: J
authors.lastname: Poznański
mesh_terms: D001665:Binding Sites; D002151:Calorimetry; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D054879:Halogenation; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013057:Spectrum Analysis; D013816:Thermodynamics; D014230:Triazoles / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008024:Ligands; D047428:Protein Kinase Inhibitors; D014230:Triazoles; C012771:benzotriazole; C570325:CSNK2A1 protein, human; D047390:Casein Kinase II