Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

• DOI: 10.1038/s41598-018-37072-x
• Publication Date: 2019-02-25T11:03:08Z
• Authors: Nan Bai; Heinrich Roder; Alex Dickson; John Karanicolas
• Keywords: Binding constant; Isothermal Titration Calorimetry; Protein ligand; Maltose-binding protein; Folding (DSP implementation)

Differential scanning fluorimetry (DSF), also known as ThermoFluor or Thermal Shift Assay, has become a commonly-used approach for detecting protein-ligand interactions, particularly in the context of fragment screening. Upon binding to folded protein, most ligands stabilize protein; thus, observing an increase temperature at which protein unfolds function ligand concentration can serve evidence direct interaction. While experimental protocols this assay are well-developed, it is not straightforward extract constants from resulting data. Because this, DSF often used probe interaction, but quantify corresponding constant (Kd). Here, we propose new analyzing Using unfolding curves varying concentrations, our "isothermal" collects these fraction that single (chosen be near transition). This greatly simplifies subsequent analysis, because circumvents complicating dependence constant; constant-temperature system then described pair coupled equilibria (protein folding/unfolding and binding/unbinding). The determined tuned, by adding chemical denaturants shift temperature. We demonstrate application isothermal analysis using data maltose maltose, two carbonic anhydrase isoforms each four inhibitors. To facilitate adoption approach, provide free easy-to-use Python program analyzes thermal implements herein ( https://sourceforge.net/projects/dsf-fitting ).