Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

Abstract

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

Discovery of Drug-Like Ligands for the Mac1 Domain of SARS-CoV-2 Nsp3

“…Such EC 50 values do not, however, describe protein–ligand affinity because DSF assays do not directly measure binding. The isothermal analysis recently described by Bai et al 9 was therefore used to estimate binding affinities. Fits of the fraction of protein unfolded at various temperatures in the presence of various ligand concentrations ( Fig.…”

“…The dissociation constant ( K d ) of Mac1 and ADPr and the equilibrium constant describing protein unfolding ( K u ) were also estimated using isothermal analysis as described by Bai et al 9 Briefly, normalized melting curves were used to calculate the fraction of protein unfolded at a particular temperature ( f u ) and those values fitted to the total ligand ( L t ) and protein ( P t ) concentrations using nonlinear regression and eq 3:…”

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

“…Thermofluor denaturation assays were performed in 7500 Fast Real-Time PCR system (Applied Biosystems) based on a previously published protocol [ 128 ]. MicroAMP Fast Reaction 8-strip tubes (Thermo Fisher, Waltham, MA, USA, P/N: 4358293) and strip caps (Thermo Fisher, P/N: 4323032) were used as reaction vessels in which 5 μM enzyme was incubated with 20 mM of appropriate buffer solution and SYPRO Orange diluted to a final 10X concentration from a 5000X stock as supplied by the manufacturer (Invitrogen, Carlsbad, CA, USA, P/N: S6650).…”

Discovery of Drug-like Ligands for the Mac1 Domain of SARS-CoV-2 Nsp3

“…The dissociation constant (Kd) of Mac1 and ADPr and the equilibrium constant describing protein unfolding (Ku) were also estimated using isothermal analysis as described by Bai et al 9 Briefly, normalized melting curves were used to calculate the fraction of protein unfolded at a particular temperature (fu), and those values fitted to the total ligand (Lt) and protein (Pt) concentrations using non-linear regression and Eq. 3:…”