Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

Abstract

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 (Hsp90αN) 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 ºC, 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 Hsp90αN-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.

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms

“…S4A, B). Notwithstanding that these DSF profiles might be reminiscent of very-slow-exchange binding [43], attempts to study the binding of compounds 1 and 3 by other methods such as surface plasmon resonance were unsuccessful (data not shown). We hypothesized that the high thermal stabilization by compounds 1 and 3 might be the result of the compounds causing further oligomerization or aggregation of TH resulting in apparent stabilization.…”

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

“…Our experience has been that careful consideration of all likely factors in the experiment at the stage of taking initial readings is essential to obtaining the best results. Furthermore, alternative theoretical treatments can reveal features of these data 15,17 . Finally, it is not uncommon for some proteins that contain natively exposed hydrophobic regions to show high background fluorescence.…”

Thermodynamics of radicicol binding to human Hsp90 alpha and beta isoforms

“…Table 2 lists the intrinsic thermodynamic parameters of radicicol binding to all tested Hsp90 protein constructs. 40 . The most precise results were obtained using the thermal shift assay.…”

“…This approach was more accurate than the displacement ITC experiments, as previously discussed. 40 The combination of ITC and TSA helps to determine ultra-tight binding constants by analyzing the pH-dependence ( Figure 6). …”

“…A 10% uncertainty in the value of unfolding enthalpy yields an uncertainty of the binding constant of ~2 fold 26 . The enthalpy of unfolding can be determined using several biophysical techniques such as DSC and acid-titration ITC 47; 48 , and by fitting raw TSA curves or ligand-dosing curves 40 . The enthalpy of unfolding was accurately measured by all these methods only for the single-domain Hsp90N, but could not be done as precisely for the full-length multidomain Hsp90 protein.…”