ITC-derived binding affinity may be biased due to titrant (nano)-aggregation...

 

Maria Winiewska, Ewa Bugajska, and Jarosław Poznański

PloS ONE
2017 vol: 12 issue: 3 pp: e0173260 doi: 10.1371/journal.pone.0173260

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.

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Topics: Solubility, Thermodynamics, Binding analysis, Isothermal titration calorimetry, Calorimetry, Protein kinases, Ultraviolet-visible spectroscopy, Enthalpy, NMR, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications

 

 

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