Measurement of nanomolar dissociation constants by titration calorimetry and thermal shift assay - radicicol binding to Hsp90 and ethoxzolamide binding to CAII.
The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90alphaN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 masculineC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90alphaN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding.
Zubrienė
Asta
A
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Matulienė
Jurgita
J
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Baranauskienė
Lina
L
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Jachno
Jelena
J
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Torresan
Jolanta
J
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Michailovienė
Vilma
V
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Cimmperman
Piotras
P
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
Matulis
Daumantas
D
Laboratory of Biothermodynamics and Drug Design / Institute of Biotechnology, Graičiūno 8, Vilnius, LT-02241, Lithuania.
D002151:Calorimetry; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism*; D005016:Ethoxzolamide / Q000737:chemistry / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D008962:Models, Theoretical; D011485:Protein Binding; D013816:Thermodynamics
D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D018942:Macrolides; D024402:Carbonic Anhydrase II; C035359:monorden; D005016:Ethoxzolamide